UNIT 6: SUBCLONING

 

INTRODUCTION:

 

One of the most common steps in a molecular cloning experiment is subcloning.  Typically one desires to move a given insert from one vector in which it is currently residing to another with more desirable properties for a given application.  For example, one might wish to move an insert from a vector in which it was expressed to another which is more useful for site directed mutagenesis.  Today many commercially available vectors are well suited for a large variety of tasks, but even so, there will be many occasions where subcloning is needed.

 

The overall sequence of a subcloning experiment is in principle quite simple.  The original vector is digested with appropriate restriction enzymes to cut out the insert.  Next the insert is purified by agarose gel electrophoresis. In the meantime, the recipient vector, which usually contains an antibiotic resistance gene,  is cut so that it is ready to receive the insert.  Finally the insert is ligated into the recipient vector, and the resulting ligation mix is used to transform competent cells, i.e., cells capable of  taking up DNA..  The cells are plated on selective media which containing an antibiotic which will kill any cells which failed to take up the vector DNA carrying an antibiotic resistance gene.  The only colonies which arise should be those containing the vector - with or without an insert. 

 

In practice, the procedure can be  more complicated (or more simple!) and many variations of  the basic scheme are used by different workers.  For example, it is not always necessary to purify the insert prior to ligation.  In some cases one can simply "shotgun" clone the restriction enzyme mix (after making sure that the restriction enzyme is removed or inactivated) by ligating the entire mix into the new vector.  This procedure can produce unwanted recombinants, such as one vector cloned into another, but since such recombinants are usually unstable and since inserts are usually smaller than vectors, they ligate into the recipient vector more efficiently than does the original  vectors.  Thus it is often possible by screening a few recombinant DNAs on agarose gels to identify a desired recombinant of the expected size. 

 

A complication often results from the fact that if  an insert is cloned into a site that has been cut with a single enzyme, the insertion can take place in either of two directions.  For some purposes this is of no consequence, but if one wishes to express a given strand of DNA, then one must arrange things so that the insertion takes place in the proper orientation.  This necessitates either using two different enzymes to create the insertion site so that insertion can take place in only a single orientation, or identifying those recombinants that by chance happen to be in the correct orientation.  Unfortunately, in most experiments, Murphy's Law seems to hold and it is easier to get the undesired recombinant!

 

Another complication is that the recipient vector may not be cut to completion.  If even a few percent of uncut vectors remain in the population, they will produce many non-recombinant colonies since often only a few percent of molecules from a typical ligation are recombinant.  Still another complication is that even if the recipient vector is cut to completion (or purified away from any uncut molecules by agarose gel electrophoresis),  the vector can recircularize in the absence of insert again giving rise to non-recombinant colonies.  We include a protocol below in which alkaline phosphatase treatment of the vector is used to block this recircularization reaction.

 

From the foregoing discussion it should be obvious that a well designed subcloning experiment needs to have several controls in order that the results be interpretable.  One can often leave out one or more of the controls we suggest, but especially for beginners it is a very good idea to include them all.  These controls are:

 

1.         Sterile Control - competent cells alone.  (Unless there is contamination, there should be no cells on this plate)

 

2.         Competency Control - competent cells plus uncut recipient vector.  (This plate should have many colonies indicating that the competent cells are capable of taking up DNA.) 

 

3.         Cut Vector Control - competent cells plus cut vector. (This plate should have very few colonies if the restriction digestion proceeded to completion.  In the real world you should probably do this control and carry out transformation before proceeding to any of the steps which follow, since if the cutting is not complete, most of the colonies you would see in an experiment will simply represent uncut vector.  If you do encounter incomplete digestion, you have the choice of finding conditions which gives more nearly complete digestion, or gel purifying the cut vector away from the uncut vector.  The latter procedure is often more trouble than it is worth.).

 

At this point the additional controls will depend on the singly or doubly cut vectors except for step 6 which common to both.

 

A. For Singly Cut Vectors

 

4.         Ligase Activity Control for singly cut vectors - competent cells plus cut vector ligated to itself.  (This plate should have many colonies if the ligase is active).

 

5.         Alkaline Phosphatase Control (see Protocol 6.5 & 6.6 for details) - Vector treated with alkaline phosphatase and then with ligase.  (This plate should have very few colonies if the phosphatase reaction was successful, since ligation can not take place.  If one is using a doubly cut vector, one need not treat with alkaline phosphatase to prevent recircularization, and this control is unnecessary - see below.  Note that if you fail to remove the phosphatase or if phosphatase or contaminating exonuclease activity actually removes bases from the cut site you will be unable to successfully ligate even with good ligase.  For this reason some workers like to include still another control when preparing the vector which is to ligate the phosphatased vector with a compatible insert which has been shown to be ligatable to a properly phosphatased vector).

 

B. For Doubly Cut Vectors:

 

4.                  Cut Vector Plus Ligase - competent cells plus cut vector plus ligase.  This control should have very few colonies since a purified doubly cut vector can not religate in the absence of insert.  If the number of colonies on this plate does increase over the control plate lacking ligase, it suggests that some fraction of the vector was not really doubly cut and that the single cut molecules are being religated.

5.                   

5.         Note that it is not necessary to do the phosphatase control.  Again you could include a known insert with compatible ends that has been shown to ligate successfully into properly doubly cut vector.

 

6.         Complete ligation reaction - competent cells plus cut vector, insert, and ligase.  This is the experiment itself and should show tens to hundreds of colonies in excess of the controls.  Note however, that if you get only a few colonies, it still may be worth screening them in case you are lucky and have successfully cloned the insert of interest.

 

PROTOCOL 6.1: DNA LIGATION WITH T4 DNA LIGASE

 

REFERENCE: MESSING ET AL., 1984; SAMBROOK ET AL., MOLECULAR CLONING, PP1.53, 1989; BRL FOCUS 2, #2-3 (1979); BRL FOCUS 8, #1 (1986) AUSUBEL ET AL., PP3.14 (1992).

 

INTRODUCTION:

 

Seven parameters affect the rate of joining of DNA fragments:  the DNA concentration, the concentration of compatible ends, the size of the fragments, the presence or absence of complementary, single-stranded ends (some nucleases form blunt ends and in these cases special techniques are needed for joining), the G + C content and length of the complementary single strands, the temperature of the reaction and ionic composition of the solution. In this section, we will consider only the case of joining of molecules having single-stranded termini (sticky-end ligation).

 

The strength of the hydrogen bonding between compatible sticky ends depends on their length and G + C content.  The melting temperature (Tm) of AT pairs is much lower than that of GC pairs.  In the cloning of EcoRI fragments of  DNA into plasmid pBR325 the sequence of each single-stranded terminus is AATT; the value of Tm for the double-stranded joint formed by annealing these ends is 4oC-5oC.  After allowing hydrogen bonds to form, the joint is covalently sealed with T4 DNA ligase, an enzyme whose activity is maximal at 37oC, a temperature at which the AATT double-stranded region would be disrupted.  Fortunately, the enzyme will function at lower temperatures, albeit slowly.  At 12oC, the double-stranded AATT joint is very unstable, but not totally so, for it is being made and broken continually.  Thus, if sufficient time is allowed at this temperature, the T4 DNA ligase succeeds in sealing the joints in the double-stranded segments that exist at a particular instant. Therefore, this temperature is usually selected as a convenient compromise between rate and efficiency.

 

The configuration of single-stranded DNA is affected by ionic conditions.  At high ionic strength single strands collapse and form intrastrand hydrogen bonds, which prevents intermolecular pairing.  At low ionic strength single strands are extended, but electrostatic repulsion between the highly charged phosphate groups prevents the strands from coming into contact.  Furthermore, owing to more subtle effects on the shape of double-stranded DNA, intramolecular joining is facilitated by high ionic strength.  In some experiments, both intermolecular  and intramolecular joining may be needed, the former to join different linear fragments and the latter for circularization of DNA prior to transformation.  In the procedures used in this manual, moderately low concentrations of monovalent and divalent cations - 50mM and 10mM, respectively - are used.  At these concentrations single strands are somewhat extended yet the charge is neutralized sufficiently to allow approach of the strands.

 

T4 DNA ligase will catalyze the ligation of either sticky or blunt ended DNA molecules with molecules of appropriate ends, but the reaction conditions under which the ligation optimally takes place are quite different depending on which type of end is being ligated.  Unfortunately there is a wide disagreement in the literature as to how much ligase is needed,  which buffer components to use and in what concentration.  Use of a vast excess of ligase may actually be counterproductive as well as wasteful since the enzyme may be contaminated by trace amounts of nucleases which can damage the sticky ends and thus inhibit ligation. Some researchers advocate using a buffer which unlike the buffer in the protocol given here lacks ATP.  This makes it easy to add whatever ATP concentration one wishes depending on whether one is doing blunt end or sticky end ligation.  This is a useful point since many workers use a higher ATP concentration (1-5 mM) and/or add spermidine to 1 mM final concentration, particularly for sticky end ligations.  High ATP and spermidine are inhibitory to blunt end ligation.  The reason for this inhibition is unknown.

 

A.  STICKY END LIGATION

 

For ligations with sticky ends that are very AT rich (e.g., Eco RI) it may be desirable to lower the temperature slightly to 12oC -15oC, instead of 16oC.  Whether or not ATP concentrations higher than 1 mM aid sticky end ligations is not clear.  Blunt end ligations are essentially the same except that the amount of ligase in the reaction is 1-2 units rather than 0.1 units and the ATP concentration is 0.5 mM instead of 1 mM.

 

PROCEDURE:

 

1.   Mix together vector and insert DNA at a 1:3 molar ratio of ends to achieve a total of 0.2 mg DNA.

 

3.         Add:

 

2 ml 10x Ligase Buffer

H2O to 20 ml final

0.1 Units T4 DNA Ligase

 

3.         Incubate at 16oC overnight.

 

4.         Transform E. coli using Protocol 6.2.  Some workers recommend diluting the reaction mixes at least 5 fold prior to transformation as some components of the reaction can inhibit transformation at high concentration.  If competent cells are not limited, we recommend transforming bacterial cells with both undiluted and diluted ligation mixtures.

 

 

 

 

MATERIALS

 

1.     1 M Tris, 7.6

 

        Compound                                                                                        Amount/ ml

        deionized H2O                                                                                           800 ml

        Tris base                                                                                                    121.1 g

 

·         Adjust to desired pH by adding concentrated HCl (~ 50 ml) and bring to volume.

·         Allow the temperature to cool to room temperature before making the final adjustments to the pH.

 

2.     1 M MgCl2 (FW=203.3)

 

        Compound                                                                                        Amount/ 1000 ml

        deionized H2O                                                                                           800 ml

        MgCl2 • 6H2O                                                                                           203.3 g

        Dissolve and adjust to 1 L

 

·         Dispense into aliquots and sterilize by autoclaving.

·         Note:  MgCl2 is extremely hygroscopic.  Buy small bottles (e.g., 100 g) and do not store opened bottles for long periods of time.

 

3.     1 M Dithiotreitol (DTT, FW=154.3)

 

        Compound                                                                                        Amount/ 20 ml

        Dithiotreitol                                                                                               3.09 g

       

·         Sterilize by filtration.

·         Dispense into 1-ml aliquots and store -20oC.

·         Note:  Do not autoclave DTT or solutions containing DDT.

·          

4.     10X Ligation Buffer

 

        Final Concentration                         Stock Solution                      Amount/ 10 ml            Amount/ 50 ml

        0.5 M Tris, pH 7.6                             1 M                                             5.0 ml                              25 ml

        100 mM MgCl2                                  1 M                                             1.0 ml                                5 ml

        100 mM Dithiothreitol                     1 M                                             1.0 ml                                5 ml

        500 mg/ml Bovine Serum                                                                     5 mg                                25 mg

           Albumin, Fraction V, Sigma (optional)

        Sterile deionized H2O to volume

 

5.     T4 DNA Ligase

 

6.     16oC water bath

 

7.     Sterile dH2O

 

B. BLUNT END LIGATION WITH T4 DNA LIGASE

 

REFERENCE: SAMBROOK ET AL. PP 1.53FF.  SEE ALSO BRL FOCUS 8, #1 (1986).

 

PROCEDURE:

 

1.   Mix together vector and insert DNA at a 1:3 molar ratio of ends to achieve a total of 0.2 mg DNA.

 

2.   Add:

 

2 ml 10x Ligase Buffer

H2O to 20 ml final

2 Units T4 DNA Ligase

 

      Incubate at 20oC-26oC  for 2-4 hours.

 

3.   Transform E. coli using Protocol 6.2.  Some workers recommend diluting the reaction mixes at least 5 fold prior to transformation as some components of the reaction can inhibit transformation at high concentration.  We are unsure whether dilution prior to transformation is better, but generally do not dilute the ligations.

 

      NOTE:  The results of this protocol vary according to DNA purity, age of ligase, and the integrity of the blunt ends.  Usual efficiencies are 5 x 104 transformants per mg of vector with a recombination efficiency of 10% assuming competent cells with an efficiency of about 5 x 105 colonies/mg of uncut DNA.

 

      Pheiffer and Zimmerman, Nucleic Acids Res. 11: 7853 (1983), have reported a dramatic improvement in efficiency of blunt end ligation in the presence of 15% PEG 8000.  Without PEG 8000 maximum ligation was achieved with <0.5 units of ligase; the efficiency was approximately 60%.  In the presence of 15% PEG 8000, 100% ligation was observed even with the smallest amount of enzyme tried (0.1 unit). 

 

Also note that although we and others recommend running an aliquot of any ligation reaction on a minigel to follow the conversion of low molecular weight fragments to higher molecular weight forms, it is unfortunately not true that maximum production of high molecular weight forms reflects the attainment of the maximum transformation efficiency-at least for blunt ended ligations.  Therefore for optimum results one should probably use the transformation frequency, not a minigel, to determine optimum conditions. (See the issue of Focus cited.)

 

MATERIALS:

 

1.     T4 DNA Ligase

 

2.     1.0 or 5.0 mM ATP

 

3.     dH2O

 

4.     100 mM Adenosine Triphosphate (ATP, FW=491.2)

 

        Compound                                                                                        Amount/1 ml               Amount/10 ml

        dH2O                                                                                                          800 ml                              8 ml

        ATP                                                                                                            60 mg                              600 mg

        Dissolve and adjust to pH 7.0 with 0.1 N NaOH

        dH2O to                                                                                                      1 ml                                 10 ml

 

·         Dispense the solution into small aliquots and store at -70oC.

 

5.     Spermidine (50 mM)

 

        Compound                                                                                        Amount/ 10 ml

        Spermidine Trichloride (FW 254.63)                                                      127.5 mg

        deionized H2O                                                                                           10 ml

       

·         Mix in sterile tube, aliquot and store at -20oC.

 

6.     Hexaminecolbalt chloride (10 mM, FW=267.5)

 

        Compound                                                                                        Amount/ 10 ml

        deionized H2O                                                                                           10 ml

        Hexaminecolbalt chloride, Co(NH3)6Cl3                                                26.8 mg

       

·         Dissolve and filter sterilize

 

7.     10X Blunt-End Ligation Buffer

        Final Concentration                         Stock Solution                      Amount/ 10 ml            Amount/ 50 ml

        0.66 M Tris, pH 7.6                           1 M                                             6.6 ml                              33 ml

           50 mM MgCl2                                 1 M                                             0.5 ml                              2.5 ml

           50 mM Dithiothreitol                    1 M                                             0.5 ml                              2.5 ml

        1 mg/ml Bovine Serum                                                                         10 mg                              50 mg

           Albumin, Fraction V, Sigma (optional)

        10 mM hexamminecolbalt chloride                                                    26.8 mg                           134 mg

          2 mM ATP                                       100 mM                                      0.2 ml                              1 ml

          5 mM Spermidine                             50 mM                                      1.0 ml                              5 ml

·         Sterile deionized H2O to volume.  Store in small aliquot at -20oC.

GENERAL INTRODUCTION TO TRANSFORMATION

 

REFERENCES: LIU, H. AND RASHIDBAIGI, A., BIOTECHNIQUES 8: 21-25 (1989); HUFF, J.P. ET AL., BIOTECHNIQUES 9: 570-577 (1990)

 

Transformation is the alteration of the genotype of a cell by the direct introduction of DNA into the cell.  The cells which are capable of taking up DNA are said to be competent.  The process of making cells competent is quite mysterious and the procedures that work for one organism often fail to work well for even closely related strains.   In what follows we shall discuss only procedures used on E. coli. 

 

Because most procedures for achieving competence involve treating cells with agents which in one way or another probably damage cell membranes, it is likely that at a minimum competence involves inducing such damage.  However, the process is certainly more complicated than just "punching holes" in the cell membrane.  The exact physiological state of the cells plays an important role in achieving competence.  Furthermore, only very few cells in a population (no more than 1 in a 100 even with the best procedures) ever achieve competence.  While cells with limited competence are satisfactory for routine subcloning of DNA when the amount of DNA is not limiting, highly competent cells are needed for constructing libraries, when only a few precious micrograms of DNA are available, and one needs to create a library containing as many primary recombinants as possible.

 

Protocols 6.2 and 6.3 present several methods for making competent cells and carrying out transformation.  The "classical" CaCl2 technique is not very efficient (103-105 transformants/mg) and most workers find the cells lose efficiency after freezing.  Typical protocols of this type are found in all the standard manuals.  Note that transformation efficiencies are usually quoted as colonies/mg of plasmid, however, competent cells are easily saturated so that addition of more than 1 ng of DNA per 100 ml of competent cells leads proportionately less efficient transformation.  Thus the transformation efficiencies stated here are calculated efficiencies and in actuality are determined using 100 pg of uncut purified plasmid DNA per 100 ml of competent cells.  Further, note that plasmids much larger than 3 kb typically transform with lower efficiency and that transformation frequency can depend on the purity of the DNA.  For all these reasons claims of extremely high transformation efficiency need to be critically evaluated.

 

A very simple procedure for making competent cells that can be frozen is due to Chung et al., Proc. Nat. Acad. Sci. USA 86: 2172-2175 (Protocol 6.2).  We have used this procedure extensively, but usually achieve only about 10%-40% of the claimed efficiency of >5 X107 transformants/mg.  However even at this level of efficiency this procedure is superior to the classical CaCl2 technique in efficiency and convenience.

 

The high efficiency procedure of Hanahan is widely quoted, but many workers fail to attain the high efficiency claimed (>108 transformants/mg), and the procedure uses a witch's brew of exotic reagents e.g., rubidium chloride and hexaminecobalt chloride.  A more recent modification to this technique has greatly simplified the composition of the solutions used (Inoue, H., Nojima, H. and Okayama, H. Gene 96: 23-28).  These authors advise growing the cells at 18oC and show that if this is done, the degree of competence is not terribly dependent on the exact density of the cells as the time of harvest.  In contrast, cells grown at 37oC are only maximally competent if harvested precisely at a specific spot in the growth curve.  It is likely that the importance of low temperature growth for achieving maximum competence involves differences in the fatty acid composition in the cell membrane at different temperatures.   The paper of Inoue et al. gives several other modifications and simplifications of the original Hanahan procedure, and it is likely that for the present it is the last word on making highly competent cells (>109 transformants/mg).

 

Finally note that electroporation which involves introduction of DNA into cells with a transient pulse of high voltage is perhaps the most efficient technique available (up to 1010 transformants/mg is claimed).  The cost of commercial capacitor banks used in this procedure has dropped, and electroporation is becoming more popular.  However, the technique still suffers from the fact that salts and other chemicals present in ligation mixtures can reduce the efficiency, so that purification of the ligated DNA is often necessary.  This can lead to losses of material, and for multiple samples is an inconvenience.  Note also that many bacterial strains that transform efficiently by electroporation may not transform well at all by other methods and vice versa.

 

PROTOCOL 6.2: ONE-STEP PEG TRANSFORMATION PROCEDURE

 

REFERENCE: CHUNG, C.T. ET AL., PROC. NATL. ACAD. SCI. USA 86: 2172-2175 (1989) CHUNG ET AL., METHODS IN ENZYMOLOGY 218: 621 (1993)

 

This method of transformation is very fast and easy and the cells can be readily frozen for later use.  As in most methods of transformation the exact mechanism by which the procedure works is unclear.  However, DMSO is a well known agent for permeabilizing many cell types, so it’s utility in aiding transformation is perhaps not surprising. 

 

PREPARATION OF COMPETENT CELLS

 

1.         A fresh overnight culture of bacteria is diluted 1:100 into prewarmed LB broth and the cells are incubated at 37oC with shaking (225 rpm) to an OD600 0.3-0.4.

 

2.         Spin cells down at moderate speed in a clinical centrifuge.  Do not pellet the cells too hard or you will find it difficult to resuspend them without vortexing which may lower transformation efficiency.  Resuspend in 1/10 volume of  ice-cold TSS buffer and mix gently. 

 

3.         For long term storage, cells are frozen immediately in a dry ice/ethanol bath and stored at -70oC.  Keeping the cells ice cold at all times before freezing increases the competency of the stored cells.  Therefore, all tubed used for freezing the cells should be prechilled on ice.  Also, when aliquotting the cells both the but the cells were resususpended in and the tubes used for the aliquots should remain on ice with the transfer taking place as quickly as possible.  Occasionally swirl the cells in the resuspension tube to keep them evenly dispersed while aliquotting.  We typically freeze the cells in tow aliquot sizes, 0.25 and 0.5 ml.

 

TRANSFORMATION

 

1.         For transformation, a 0.1 ml aliquot of cells is pipetted into a cold polypropylene tube (Falcon 2059) containing 1 ml (100 pg) of plasmid DNA and the cell/DNA suspension is mixed gently.  (When frozen cells are used, cells are thawed slowly on ice and used immediately.)

 

2.         The cell/DNA mixture is incubated  on ice for 60 min.  Shorter incubation times probably work well as well.

 

3.         A 0.9 ml aliquot of  LB broth (pH 6.5) containing 20 mM glucose is added, and the cells are incubated at 37oC with shaking (225 rpm) for 1 hour to allow expression of the antibiotic-resistance gene.

 

 

 

4.         Plate 100 ml of cells.  If you have reason to believe the transformation efficiency will be low, spin the samples in a microfuge for 1 min.  Discard 0.8 ml of the supernatant.  Make sure to leave 50-100 ml in the tube.  Resuspend the pellet in the remaining supernatant.  Spread 100 ml on 100 mm2 LB agar plates containing 50 mg/ml ampicillin or other appropriate antibiotic.

 

            Note:  The number of colonies found on the plate containing the 9X cell concentration may not be as high as one would predict from the number colonies on the 1X plate.  This is because substances leaching from the dying cells on the 9X plate can be inhibitory to the growth of the transformed cells.

 

NOTES: Miller (personal communication) states that good results are dependent on proper preparation of the TSS.  The easiest way to make TSS is to autoclave the LB, add the Mg, DMSO, and PEG, dissolve these components and filter sterilize the TSS through a nylon filter.  Nylon is used because DMSO will dissolve many types of filters.

           

            Alternatively the non-sterile components can be sterilized and added sterilely to the LB.  The components are sterilized as follows:  Mg- autoclave or filter sterilize; DMSO-filter sterilize through nylon or use a new bottle and keep sterile, and 60% PEG filter sterilize.  One thing to avoid is mixing everything together and autoclaving the solution. 

 

Use the highest quality and freshest, reagents available.  We seem to get better results using Sigma's molecular biology grade of PEG.  Miller recommends using Fluka DMSO.  Using the Sigma PEG and  several different bottles of reagent grade DMSO we have obtained efficiencies about 10%-40% of those claimed.

 

Note that according to the authors it is not necessary to heat shock the cells in this procedure, although we have on occasion found it does help.  Finally note that the transformation efficiency is measured with 100 pg of uncut plasmid DNA.  If the cells are prepared properly, the efficiency will be nearly as high using 10 ng of DNA.  Beyond 10 ng transformation efficiency does not usually increase linearly with proportionate increases in DNA concentration.  Hence, the estimates of competency will also decline for DNA concentrations beyond a certain point.

 

MATERIALS:

 

1.     LB Medium (Luria-Bertani Medium, pH 6.5)

       

        In a clean dry 2L Erlenmeyer flask add the constituents listed below.  If you turn the Bacto-tryptone and Yeast extract bottles on their side and gently tap them to loosen up the contents before you open the bottle, the contents will shift so they can be more easily scooped out of the bottle without generating a large amount of dust.

 

        Compound                                                                                        Amount/1000 ml

        Bacto-tryptone                                                                                             10 g

        Yeast extract                                                                                                   5 g        

         NaCl                                                                                                              10 g

 

        Add H2O to 800 ml. Stir until the solutes have dissolved.  Adjust to pH 6.5 with 5 N NaOH.  Note: Be sure to adjust the pH to 6.5 not 7.5 as normally done with LB.

 

2. TSS is LB broth pH 6.5 with:

 

        Final Concentration                         Stock Solution                      Amount/ 100 ml

        deionized H2O                                                                                       80 ml

        10% PEG-molecular                               PEG                                        10 g

              weight 3350 or 8000

         5 % DMSO,                                           DMSO                                     0.5 ml

        Dimethylsulfoxide

        molecular biology grade

20 to 50 mM Mg2+                          1 M                                       2 to 5 ml

MgSO4 or MgCl2

at a final pH of 6.5.

 

·         Deionized H2O to volume.

 

·         See notes in the text above on how to properly make this solution.  Making this solution properly is one of the most important factors determining the efficiency of the cells.

PROTOCOL 6.3: HIGH EFFICIENCY TRANSFORMATION OF E. coli WITH PLASMIDS

 

REFERENCE: INOUE, H. NOJIMA, H. AND OKAYAMA, H. (1990) GENE 96:23-28.

 

INTRODUCTION:

 

This is a good general protocol for producing competent cells.  It takes longer to do than the PEG procedure (Protocol 6.2), but is still relatively easy compared to the Hanahan procedure. The most unusual step in the procedure is that the cells are grown at 18 0 C.  This low temperature presumably modifies the lipid composition of the cell membrane resulting in a more efficient uptake of DNA.  The cells are usually in the 1 X 107 colonies/mg range or better.  If you require extremely competent cells (1 X 109 colonies/mg range) for tasks such as library construction, it is best to buy commercially prepared cells.

 

Preparation of Competent Cells

 

1.         Streak frozen cells on LB agar plate and grow overnight at 37oC. 

 

2.         Isolate about ten to twelve large (2-3 mm) colonies with a plastic loop and inoculate 250 ml of SOB medium in a 2 liter flask, grow to A600 of 0.6 at 18oC, with vigorous shaking (200-250 rpm).

 

3.         Remove flask from incubator and place on ice for 10 min.

 

4.         Transfer culture to centrifuge bottles and spin at 2500 X g (3000 rpm) for 20 min. at 4oC. 

 

5.         Resuspend pellets in 80 ml of ice-cold TB, incubate in an ice bath for 20 min. and spin down as above.

 

6.         Gently resuspend cells in 20 ml of ice cold TB

 

7.         Add DMSO with gentle swirling to a final concentration of 7%

 

8.         Incubate in ice bath for 10 min.

 

9.         Dispense in 1 to 2 ml aliquots and freeze in liquid nitrogen.

 

10.       Store -80oC for several months.

 

 

 

 

Transformation

 

1.         Thaw competent cells  at room temperature.

 

2.         Dispense 200 ml aliquots into 15 ml polypropylene tubes and place in ice bath.  Recent evidence suggests that if one adds sufficient b-mercaptoethanol or DTT to make each aliquot 20 mM final concentration, prior to adding the DNA, that transformation efficiencies go up 5 to 20 fold.

 

3.         Add 1 - 5 ml of the ligation mix to each tube.  We suggest you start with 1 ml because ligation mixtures contain compounds that often dramatically lower transformation efficiencies.  Hence less of the ligation mixture will often yield more transfomants.

 

4.         Incubate cells in ice bath for 30 min.  You may wish to gently swirl cells once or twice during this period and quickly return the tube to the ice bucket.

 

5.         Heat pulse without agitation at 42oC for 30 sec and transfer to an ice bath.

 

6.         Add 0.8 ml of SOC and grow at 37oC shaking vigorously for 1 hr.

 

7.         Plate 100 ml of cells.  If you have reason to believe the transformation efficiency will be low, spin the samples in a microfuge for 1 min.  Discard 0.8 ml of the supernatant.  Make sure to leave 50-100 ml in the tube.  Resuspend the pellet in the remaining supernatant.  Spread 100 ml on 100 mm2 LB agar plates containing 50 mg/ml ampicillin or other appropriate antibiotic.

 

            Note:  The number of colonies found on the plate containing the 9X cell concentration may not be as high as one would predict from the number colonies on the 1X plate.  This is because substances leaching from the dying cells on the 9X plate can be inhibitory to the growth of the transformed cells.

 

8.         If you are using blue/white color selection, first spread 30 ml of 2% XGAL in N,N-DMF and 30 ml 20 mM IPTG in water over the plate with a spreader.

 

9.         Spread cells on LB plates continuing the appropriate antibiotic.

 

MATERIALS:

 

1.     TB Buffer

 

        Compound                                    MW                                  Amount/ 1000 ml

        10 mM    Pipes                              302.40                                     3.02 g

        55 mM    MnCl2                             197.91                                     10.89 g

        15 mM   CaCl2                               147.02                                     2.21 g

        250 mM  KCl                                 74.55                                       18.64 g

 

·         Mix all components except MnCl2 and adjust pH to 6.7 with KOH. 

·         MnCl2 is then added and the solution filter sterilized through a prerinsed 0.45 mm filter unit and stored at 4oC.

 

2.     SOB Medium

 

        Compound                                                                                        Amount/ 1000 ml

        Deionized H2O                                                                                          950 ml

        Bacto-tryptone                                                                                         20 g

        Bacto-yeast extract                                                                                    5 g

        NaCl                                                                                                           0.5 g

 

·         Shake until the solutes have dissolved.

·         Add 10 ml of 250 mm solution of KCl adjusted to pH 7.0. (~ 0.2 ml)

·         Deionized H2O to 1 liter

·         Sterilize by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle.

·         Just before use, add 5 ml of a sterile solution of 2 M MgCl2

·         Adjust pH to 7.5 with potassium or sodium hydroxide and sterilize by autoclaving.  Just before use, add 20 ml of 1 M MgSO4, sterilized separately by autoclaving.

 

3.     250 mM KCL

 

        Compound                                                                                        Amount/ 100 ml

        KCL                                                                                                            1.86 g

        Adjust the pH to 7.0 with 5 N NaOH - ~0.2 ml

        Bring to volume with deionized water.

 

4.     2 M MgCl2

 

        Compound                                                                                        Amount/ 100 ml

        Deionized H2O                                                                                          90 ml

        MgCl2                                                                                                         19 g

 

 

5.     SOC Medium

        Compound                                                                                        Amount/ 1000 ml       

        Bacto-tryptone                                                                                           20    g

        Bacto-yeast extract                                                                                      5    g

        NaCl                                                                                                               0.5 g

        Deionized H2O                                                                                          950    ml

 

·         Shake until the solutes have dissolved.

·         Add 10 ml of 250 mM KCl                                                              

·         Adjust the pH to 7.0 with 5 N NaOH (~ 0.2 ml)

·         Deionized H20 to 1 liter

·         Sterilize by autoclaving for 20 minutes at 15 lb/sq. in. on liquid cycle.

·         Allow solution to cool to 60oC or less

·         Add 20 ml of sterile 1M glucose                                                   

·         Just before use, add 5 ml of a sterile solution of 2 M MgCl2

PROTOCOL 6.4:  TRANSFORMATION Of E. COLI COMPETENT CELLS PREPARED BY NOVAGEN.

 

A number of companies (e.g. Gibco/BRL, Novagen, Stratagene, Etc.) sell competent cells.  The higher competency cells sold by these companies are generally better than you can prepare in the laboratory and should be considered if your application calls for high cloning efficiency.  Every time you receive a new batch of cells you should  check the competency with one to 100 pg of supercoiled plasmid DNA to make sure they are as competent as the company is claiming.  The advantage of these cells is that they are efficient and convenient.  The drawback is that they are expensive, particularly for routine subcloning.

 

The following protocol is for transformation of NovaBlue competent cells.  The protocol varies slightly at the beginning depending on what volume of competent cells you wish to transform.  For maximum efficiency, the sample DNA must be free of phenol, ethanol, protein, and detergents.  It should be dissolved in TE buffer (10 mM Tris-HCl, pH 7.8, 1 mM EDTA) or in water.  Efficiencies should be linear up to about 10 ng plasmid in 100 ml transformation. 

 

A.  Transformation using 20 ml of competent cells:

 

1.         Thaw the required number of tubes of cells on ice.  Pre-chill the required number of 1.5 ml polypropylene microcentrifuge tubes on ice.

 

2.         Resuspend cells by finger flicking once or twice just prior to pipetting 20 ml aliquots of cells into the pre-chilled microfuge tubes.  Failure to resuspend the cells will lead to poor transformation results.  Also do the pipetting as quickly as possible to keep the cells cold.  Keeping the cells cold enhances transformation efficiency.

 

3.         Add 1 ml of 20 ml ligation mixture.

 

            (Optional) To determine transformation efficiency, add 1 ml, containing 100 pg test plasmid for 50 ml transformation.  Gently flick the tube to mix.

 

4.         Proceed to step 6 below

 

B.  If using 50 ml of competent cells start at this point:

 

1.         Thaw the required number of tubes of cells on ice and mix gently to assure that the cells are evenly suspended.  Pre-chill the required number of 1.5 ml polypropylene microcentrifuge tubes on ice.

 

2.         Resuspend cells by finger flicking once or twice just prior to pipetting 50 ml aliquots of cells into the pre-chilled polypropylene tubes (Falcon 2059).  Failure to resuspend the cells will lead to poor transformation results.  Also do the pipetting as quickly as possible to keep the cells cold.  Keeping the cells cold enhances transformation efficiency.

 

3.         (Optional) To determine transformation efficiency, add 1 ml, containing 100 pg test plasmid for 50 ml transformation.  Gently flick the tube to mix.

 

4.         For DNA from ligation reaction, add 1 ml or less of the ligation directly to the cells.  For recombinants, expect 103 - 107 transformants/mg plasmid, depending on the particular insert and the ligation efficiency. Note: Transformation efficiencies can be increased several fold by diluting the ligation reaction 5-fold with TE or water prior to adding the DNA, or by extracting the ligation reaction twice with 1:1 TE-buffered phenol: CIAA (24:1 chloroform: isoamyl alcohol), once with CIAA, precipitating in the presence of NaOAc, and resuspending in TE or water before adding the DNA to cells.  Gently flick the tube to mix.

 

5.         Go to step 6 below.

 

C.  If using 100 ml of competent cells start at this point:

 

1.         Thaw the required number of tubes of cells on ice and mix gently to assure that the cells are evenly suspended.  Pre-chill the required number of 17 X 100 mm polypropylene tubes (Falcon 2059) on ice.

 

2.         Resuspend cells by finger flicking once or twice just prior to pipetting 100 ml aliquots of cells into the pre-chilled polypropylene tubes (Falcon 2059).  Failure to resuspend the cells will lead to poor transformation results.  Also do the pipetting as quickly as possible to keep the cells cold.  Keeping the cells cold enhances transformation efficiency.

 

3.         (Optional) To determine transformation efficiency add 100 pg test plasmid per 100 ml transformation.  Gently flick the tube and return it to ice, or gently spin the tube once or twice while it is on ice, to disperse the DNA throughout the cell suspension.

 

4.         For DNA from ligation reaction, add 2 ml or less of the ligation directly to the cells.  For recombinants, expect 103 - 107 transformants/mg plasmid, depending on the particular insert and the ligation efficiency.  Gently flick the tube to mix.

 

5.         Proceed to step 6 below

 

 

 

 

Common Steps For A, B and C.

 

6.         Place the tubes on ice for 30 minutes.

 

7.         Heat the tubes for exactly 45 seconds (40 seconds for 20 ml transformation) in a 42°C water bath; do not shake.

 

8.         Place on ice for 2 minutes.

 

9.         Add 0.9 ml of room temperature SOC or LB medium unless doing the 20 ml transformation in which case add 80 ml of SOC or LB

 

10.       Shake at 200-250 rpm at 37°C for 1 hour.

 

11.       Plate 100 ml of cells (50 ml of the 20 ml transformation).  If you have reason to believe the transformation efficiency will be low, spin the samples in a microfuge for 1 min.  Discard 0.8 ml of the supernatant.  Make sure to leave 50-100 ml in the tube.  Resuspend the pellet in the remaining supernatant.  Spread 100 ml on 100 mm2 LB agar plates containing 50 mg/ml ampicillin or other appropriate antibiotic.

 

            Note:  The number of colonies found on the plate containing the 9X cell concentration may not be as high as one would predict from the number colonies on the 1X plate.  This is because substances leaching from the dying cells on the 9X plate can be inhibitory to the growth of the transformed cells.

 

11.       Incubate plates overnight at 37°C.

 

MATERIALS:

 

1.     Competent Cells

 

2.     37oC and 42oC water baths

 

3.     SOC MEDIUM:

 

Component                                     Final Concentration                             Amount per 100 ml

 

Bacto-tryptone                               2%                                                          2.0 g

Yeast Extract                                   0.5%                                                       0.5 g

NaCl                                                 10 mM                                                    1 ml 1M NaCl

KCl                                                   2.5 mM                                                   0.25 Ml 1M KCl

MgCl2, MgSO4                                20 mM (10mM each)                            1 ml 2M Mg Stock (1M each)

Glucose                                            20 mM                                                    1 ml 2M glucose

Deionized H20                                                                                                 to 100 ml

 

Dissolve bacto-tryptone, yeast extract, NaCl and KCl in H20 and bring to 98 ml.  Autoclave and cool to room temperature.  Add Mg stock (1M, MgCl2 6 H20, 1M MgSO4 7 H20 : filter-sterilized) and glucose (filter-sterilized).  The pH should be between 6.9 and 7.1.  Note: Filter-sterilizing units should be prerinsed with deionized H20 before use to remove any toxic material from the filter.

 

PROTOCOL 6.5:  DEPHOSPHORYLATION OF DNA

 

REFERENCE: SAMBROOK ET AL., MOLECULAR CLONING, PP. 1.60

 

INTRODUCTION:

 

In many cloning experiments one of the most common sources of difficulty is the recircularization  of the vector during ligation  without insertion of any cloned DNA.  Unless one has a rapid assay for the insertional inactivation of the gene at the site of insertion e.g. a change of color on an indicator plate, or unless one has a strong selection against the religated vector e.g. by the cI/hflA selection used when cloning into l gt10 or unless one is using a doubly cut vector which can not recircularize due to incompatible ends, many of the cells which take up the vector will have no DNA inserts.  One way to prevent this unhappy occurrence is to treat the cut vector with a phosphatase which removes the 5’ phosphate group from the vector.  Since ligation reactions have an absolute requirement for the presence of a 5’ phosphate, this treatment effectively blocks the religation of the vector as the required phosphate group can only come from the DNA insert.

 

The terminal 5' phosphates can be removed from DNA by treatment either with bacterial alkaline phosphatase (BAP) or with calf intestinal alkaline phosphatase (CIP). The latter enzyme has the considerable advantage in that following the phosphatase reaction it can be completely inactivated by heating to 68oC in SDS.  BAP, on the other hand, is heat-resistant (in fact, reactions with BAP are usually carried out at 68oC to  suppress the activity of an exonuclease that often contaminates preparations of the enzyme). Either multiple extractions with phenol/chloroform or purification of the DNA fragment by gel electrophoresis are required to remove all traces of BAP activity.  For most purposes, therefore, CIP is the preferred enzyme although a third alkaline phosphatase from Arctic shrimp that is very heat labile is also becoming popular because it is so easy to inactivate following the dephosphorylation reaction.  Although we give below a "classical" protocol  for dephosphorylations,  many  workers claim to obtain excellent results simply by adding  alkaline phosphatase to restriction enzyme digestions during the digestion.  In this case, the buffer can hardly be optimum, but apparently enough dephosphorylation occurs anyway to greatly reduce the recircularization problem.

 

PROCEDURE:

 

1.   Digest the DNA to completion with the restriction enzyme of choice.  Extract once with phenol/chloroform and precipitate the DNA with ethanol.  Remember to save some undigested DNA for a control.

 

2.   Dissolve the DNA in a minimum volume of 10mM Tris-HCl, pH 8.0.  Add:

 

10x CIP buffer    5 ml

water     to 48 ml

CIP       (see amount below).

 

Remember to save some cut  DNA that you do not dephosphorylate for a control.

 

a.         0.01 units of CIP are needed to remove the terminal phosphates from 1 pmole of 5' ends of DNA (1 pmole of 5' ends of a 4-kb linear DNA is 1.6 mg).

 

b.         To dephosphorylate protruding 5' termini, add CIP as in "a", incubate at 37oC for 30 minutes, add a second aliquot of CIP, and continue the incubation for a further 30 minutes.

 

c.         To dephosphorylate DNA with blunt ends or recessed 5' termini, add CIP as in step "a", incubate for 15 minutes at 37oC followed by a second incubation for 15 minutes at 56oC. Then add a second aliquot of CIP and repeat the incubations at both temperatures.

 

3.         Add 40 ml of water, 10 ml of 10x STE, and 5 ml of 10% SDS.  Heat to 68oC for 15 minutes.

 

4.         Extract twice with phenol/chloroform and twice with chloroform.  (Some protocols add a proteinase K digestion to the phenol extraction step.)

 

5.         Pass the DNA through a Sephadex G-50 spin column. (This step is used to remove the excess ammonium sulfate which was once used to store CIP.  If using CIP lacking ammonium sulfate, this step can be omitted.)

 

6.         Precipitate the DNA with ethanol.  It is now ready for ligation or kinasing.

 

Notes:  Although the above procedures are the recommended way to dephosphorylate a vector, some workers simply add the alkaline phosphatase to the restriction enzyme digestions and carry out restriction and dephosphorylation simultaneously.  Under such conditions the enzyme is being used far from its pH and salt optimum, so results are not guaranteed!  However, this fast method may be worth a try providing you run appropriate controls to ensure that the dephosphorylation was successful.

 

MATERIALS:

 

10x CIP buffer

 

          0.5 M Tris-Cl, pH 9.0

          10 mM MgCl2

          10 mM ZnCl2

          10 mM spermidine (optional)

 

 

10x STE

 

          100 mM Tris-Cl, pH 8.0

          1M NaCl

          10mM EDTA

 

PROTOCOL 6.6: DEPHOSPHORYLATION OF DNA USING SHRIMP ALKALINE PHOSPHATASE

SOURCE:  USB Molecular Biology Reagents/Protocol Book

 

INTRODUCTION:

 

Alkaline phosphatase, isolated from Arctic shrimp, has approximately the same specific activity as the calf intestine enzyme (800-1000 units/mg at 25oC, pH 9.6) but, unlike the calf enzyme, it is completely and irreversibly inactivated in Tris buffers at pH 8.0-8.5 by simply heating for 15 minutes at 65oC.  No further treatment is necessary.  This novel alkaline phosphatase is stable when stored at -20oC  in glycerol-containing buffer.  It is purified to apparent homogeneity and is free of all contaminating endonucleases, exonucleases, and ribonucleases. 

 

PROCEDURE:

 

The rate of removal of the terminal 5' phosphate from double-stranded DNA depends on the structure of the terminus.  Termini with 5' protruding ends are more reactive than those with blunt ends or those with 5'-recessed ends.  The reaction rate also depends on the temperature and magnesium concentration.  USB recommends that dephosphorylation of DNA be done at 37oC in the following buffer.

 

20 mM Tris-HCl pH 8.0

10mM MgCl2

20 mg/ml DNA

 

The amount of enzyme depends on the kind of termini and the amount of DNA.  For a typical reaction using 1.0 pmol of DNA termini (2.5 mg of 3Kb plasmid), the following amounts were found to be effective.

 

Terminus                      Units of phosphatase (1 hour, 37oC )

5’-Protruding                    0.1 units

Blunt                                 0.2 units

5’-Recessed                      0.5 units

 

Note:  These are minimum effective amounts.  It may be prudent to use more enzyme or longer incubation times to assure complete dephosphorylation.