DNA extract

DNA extract kdorfman Mon, 03/12/2012 - 15:12

Isolating, digest out the P-element, Ligate P-element line DNA

Use Qiagen DNeasy Blood & Tissue Kit 69504 $141.12 for 50 reactions

LiCl/KAc

LiCl/KAc kdorfman Mon, 03/12/2012 - 18:01

LiCl/KAc

1 part 5M KAc : 2.5 parts 6M LiCl

Stock solution 14 17.5 21 mL final volume
5M KAc 4 5 6 mL
6M LiCl 10 12.5 15 mL

Ligase

Ligase kdorfman Mon, 03/12/2012 - 18:03

T4 DNA Ligase

NEB M0202S

Get from New England Biolabs Freezer Program in Fernald

Store in freezer

Comes with 10X ligase buffer (includes ATP

NaOAc

NaOAc kdorfman Mon, 03/12/2012 - 18:06

3M NaOAc

PBS for DNA

PBS for DNA kdorfman Mon, 03/12/2012 - 18:04

PBS

~200 mL/prep

4 preps per group

Aliquot 1 mL per group

Soution A

Soution A kdorfman Mon, 03/12/2012 - 18:00

Solution A

100 mM Tris-HCl pH 7.5

100mM EDTA

100 mM NaCl

0.5% SDS

Stock solution 10 25 50 100 mL final volume
1 M Tris 1 2.5 5 10 mL
0.5 M EDTA 2 5 10 20 mL
5 M NaCl 0.02 0.05 0.1 0.2 mL
20% SDS 0.25 0.625 1.25 2.5 mL

Make 10 mL

pH to 7.5