{"id":2302,"date":"2017-06-08T20:53:52","date_gmt":"2017-06-09T00:53:52","guid":{"rendered":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/recipies\/"},"modified":"2018-07-20T16:38:09","modified_gmt":"2018-07-20T20:38:09","slug":"recipies","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/recipies\/","title":{"rendered":"Media Recipes"},"content":{"rendered":"
\n

A Dohlman Lab Protocol<\/em><\/p>\n

\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
2X LB (per liter)<\/span><\/b><\/td>\n<\/td>\n<\/tr>\n
water<\/td>\n1000 ml<\/td>\n<\/tr>\n
tryptone<\/td>\n20 g<\/td>\n<\/tr>\n
yeast extract<\/td>\n10 g<\/td>\n<\/tr>\n
NaCl<\/td>\n5 g<\/td>\n<\/tr>\n
pH to 7.0, (~ 52 drops 1N NaOH)<\/td>\n<\/td>\n<\/tr>\n
for plates (add a stir bar):<\/span><\/b><\/td>\n<\/td>\n<\/tr>\n
bacto-agar<\/td>\n15 g<\/td>\n<\/tr>\n
autoclave & allow to cool to the point that the glass is warm but not burning hot<\/td>\n<\/td>\n<\/tr>\n
for plasmid selection:<\/span><\/b><\/td>\n<\/td>\n<\/tr>\n
ampicillin (or carbenicillin*)
\n1000 X stock (50 mg\/ml)<\/td>\n		1 ml<\/td>\n<\/tr>\n
*more expensive but more stable, use for plates<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

LABEL YOUR PLATES USING THE APPROPRIATE COLOR CODE<\/b><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
2XLB<\/td>\nBlk<\/td>\n<\/tr>\n
2XLB carb<\/td>\nBlk\/Blu<\/td>\n<\/tr>\n
2XLB kan<\/td>\nBlk\/Grn<\/td>\n<\/tr>\n
SCD (per liter)<\/span><\/b><\/td>\n<\/td>\n<\/tr>\n
water<\/td>\n900 ml<\/td>\n<\/tr>\n
drop-out mix<\/td>\n0.940 g<\/td>\n<\/tr>\n
yeast nitrogen base w\/o amino acids and
\nw\/o ammonium sulfate<\/td>\n		1.7 g<\/td>\n<\/tr>\n
ammonium sulfate<\/td>\n5 g<\/td>\n<\/tr>\n
for plates (add a stir bar):<\/span><\/b><\/td>\n<\/td>\n<\/tr>\n
bacto-agar<\/td>\n20 g<\/td>\n<\/tr>\n
NaOH<\/td>\none pellet<\/td>\n<\/tr>\n
autoclave & allow to cool to the point that the glass is warm but not burning hot<\/td>\n<\/td>\n<\/tr>\n
\u201c40X separate mix\u201d (filter sterilized)<\/td>\n25 ml<\/td>\n<\/tr>\n
20 % dextrose (autoclaved separately)<\/td>\n100 ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

LABEL YOUR PLATES USING THE APPROPRIATE COLOR CODE<\/b><\/p>\n\n\n\n\n\n\n\n\n\n
SCD-Ura<\/td>\nRed\/Grn<\/td>\nSCD-Ura-Leu<\/td>\nRed\/Blu\/Grn<\/td>\n<\/tr>\n
SCD-Leu<\/td>\nRed\/Blu<\/td>\nSCD-Ura-Trp<\/td>\nGrn\/Blu\/Blu<\/td>\n<\/tr>\n
SCD-Trp<\/td>\nRed\/Blu\/Blu<\/td>\nSCD-Ura-His<\/td>\nGrn\/Blu\/Blk<\/td>\n<\/tr>\n
SCD-His<\/td>\nGrn\/Blk<\/td>\nSCD-Leu-Trp<\/td>\nRed\/Blu\/Blk\/Blu<\/td>\n<\/tr>\n
SCD-Ura-Leu-Trp<\/td>\nRed\/Red\/Red<\/td>\nSCD-Leu-His<\/td>\nGrn\/Grn\/Blk<\/td>\n<\/tr>\n
SCD-Ura-Trp-His<\/td>\nRed\/Grn\/Blu\/Blu<\/td>\nSCD-Trp-His<\/td>\nRed\/Red\/Blu<\/td>\n<\/tr>\n
SCD-Leu-Trp-His<\/td>\nBlk\/Red\/Blu\/Grn<\/td>\n5FOA<\/td>\nRed\/Blk\/Grn<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
YPD (per liter)<\/b><\/span><\/td>\n900 ml<\/td>\n<\/tr>\n
water<\/td>\n<\/td>\n<\/tr>\n
yeast extract<\/td>\n10 g<\/td>\n<\/tr>\n
peptone<\/td>\n20 g<\/td>\n<\/tr>\n
for plates (add a stir bar):<\/span><\/b><\/td>\n<\/td>\n<\/tr>\n
bacto-agar<\/td>\n20 g<\/td>\n<\/tr>\n
autoclave<\/b><\/td>\n<\/td>\n<\/tr>\n
20 % dextrose (autoclaved separately)<\/td>\n100 ml<\/td>\n<\/tr>\n
or<\/td>\n<\/td>\n<\/tr>\n
20 % galactose<\/td>\n100 ml<\/td>\n<\/tr>\n
20 % sucrose<\/td>\n10 ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

LABEL YOUR PLATES USING THE APPROPRIATE COLOR CODE<\/span><\/b><\/p>\n\n\n\n\n
YPD<\/td>\nRed<\/td>\n<\/tr>\n
YPG\/S<\/td>\nRed\/Red\/Blk<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Drop out mix powder<\/b><\/p>\n

Omit the appropriate amino acid for (URA3, LEU2, HIS3) plasmid selection.
\nMix the powder using a coffee grinder.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
<\/td>\nAmount (g)<\/td>\nFinal conc. (mg\/l)<\/td>\n<\/tr>\n
Arg-HCl<\/td>\n0.4<\/td>\n20<\/td>\n<\/tr>\n
His-HCl (omit for SCD-His)<\/td>\n0.4<\/td>\n20<\/td>\n<\/tr>\n
Met<\/td>\n0.4<\/td>\n20<\/td>\n<\/tr>\n
Uracil (omit for SCD-Ura)<\/td>\n0.4<\/td>\n20<\/td>\n<\/tr>\n
Ile (omit for SCD-Leu)<\/td>\n0.6<\/td>\n30<\/td>\n<\/tr>\n
Leu (omit for SCD-Leu)<\/td>\n0.6<\/td>\n30<\/td>\n<\/tr>\n
Lys-HCl<\/td>\n0.6<\/td>\n30<\/td>\n<\/tr>\n
Tyr<\/td>\n0.6<\/td>\n30<\/td>\n<\/tr>\n
Adenine-HCl<\/td>\n0.8<\/td>\n40<\/td>\n<\/tr>\n
Phe<\/td>\n1.0<\/td>\n50<\/td>\n<\/tr>\n
Glu<\/td>\n2.0<\/td>\n100<\/td>\n<\/tr>\n
Val<\/td>\n3.0<\/td>\n150<\/td>\n<\/tr>\n
Ser<\/td>\n8.0<\/td>\n400<\/td>\n<\/tr>\n
Add 0.940 g to 1 liter of media & autoclave<\/td>\n<\/td>\n<\/td>\n<\/tr>\n
40X Separate mix liquid<\/b><\/span><\/td>\n<\/td>\n<\/td>\n<\/tr>\n
<\/td>\nAmount (g)<\/td>\nFinal conc. (mg\/l)<\/td>\n<\/tr>\n
Trp (omit for SCD-Trp)<\/td>\n0.8<\/td>\n40<\/td>\n<\/tr>\n
Asp<\/td>\n2.0<\/td>\n100<\/td>\n<\/tr>\n
Thr<\/td>\n4.0<\/td>\n200<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Dissolve in 500 ml water, filter sterilize, and add 25 ml per liter of media
\n(after autoclaving & cooling)<\/p>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

A Dohlman Lab Protocol 2X LB (per liter) water 1000 ml tryptone 20 g yeast extract 10 g NaCl 5 g pH to 7.0, (~ 52 drops 1N NaOH) for plates (add a stir bar): bacto-agar 15 g autoclave & allow to cool to the point that the glass is warm but not burning hot … Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":11,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2302","page","type-page","status-publish","hentry","odd"],"acf":[],"_links_to":[],"_links_to_target":[],"_links":{"self":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2302","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/users\/22429"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/comments?post=2302"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2302\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2239"}],"wp:attachment":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/media?parent=2302"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}