Solutions for molecular cloning--1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )
Bst "short" termination "T" mix: 8 uM dTTP, 164 uM
dATP, 164 uM dCTP, 164 uM dTTP, 600 uM ddTTP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.
16.4 ul 5 mM dATP
16.4 ul 5 mM dCTP
16.4 ul 5 mM 7deaza-dGTP
8 ul 0.5 mM dTTP
60 ul 5 mM ddTTP
50 ul 50:1 TE buffer
332.8 ul sddH2O
500 ul
aliquot (18 ul for 6 reactions) and store at -70degC
Bst "long"
termination "A" mix: 8 uM dATP, 164 uM dCTP, 164 uM 7deaza-dGTP, 164 uM dTTP, 110 uM ddATP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.
8 ul 0.5 mM dATP
16.4 ul 5 mM dCTP
16.4 ul 5 mM 7deaza-dGTP
16.4 ul 5 mM dTTP
11 ul 5 mM ddATP
50 ul 50:1 TE buffer
381.8 ul sddH2O
500 ul
aliquot (18 ul for 6 reactions) and store at -70degC
Bst "long"
termination "C" mix: 8 uM dCTP, 164 uM dATP, 164 uM 7deaza-dGTP, 164 uM dTTP, 65 uM ddCTP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.
16.4 ul 5 mM dATP
8 ul 0.5 mM dCTP
16.4 ul 5 mM 7deaza-dGTP
16.4 ul 5 mM dTTP
6.5 ul 5 mM ddCTP
50 ul 50:1 TE buffer
386.3 ul sddH2O
500 ul
aliquot (18 ul for 6 reactions) and store at -70degC
Bst "long"
termination "G" mix: 8 uM dGTP, 164 uM dATP, 164 uM dCTP, 164 uM dTTP, 70 uM ddGTP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.
16.4 ul 5 mM dATP
16.4 ul 5 mM dCTP
8 ul 0.5 mM 7deaza-dGTP
16.4 ul 5 mM dTTP
7 ul 5 mM ddGTP
50 ul 50:1 TE buffer
385.8 ul sddH2O
500 ul
aliquot (18 ul for 6 reactions) and store at -70degC
Bst "long"
termination "T" mix: 8 uM dTTP, 164 uM dATP, 164 uM dCTP, 164 uM dTTP, 150 uM ddTTP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.
16.4 ul 5 mM dATP
16.4 ul 5 mM dCTP
16.4 ul 5 mM 7deaza-dGTP
8 ul 0.5 mM dTTP
15 ul 5 mM ddTTP
50 ul 50:1 TE buffer
377.8 ul sddH2O
500 ul
aliquot (18 ul for 6 reactions) and store at -70degC
100 mM
calcium chloride (CaCl2):
1.48 g CaCl2-2H2O
ddH2O to 100 ml
autoclave to sterilize (store at 4deg.C).
50 mM calcium
chloride:
0.74 g CaCl2-2H2O
ddH2O to 100 ml
autoclave to sterilize (store at 4deg.C).
Chloramphenicol 10
mg/ml stock:
2 g Chloramphenicol (Sigma C-0378)
200 ml 100% (or 95%) ethanol
Final volume 200
ml
Sterilize by filtering through a Nalgene filter (#8-0000-22 0301)
Store in the cold room in a foil wrapped screw cap Nalgene bottle
Use as an additive to microbial growth media at a final concentration of 15 ug/ml media.
For 200 ml media, add 300 ul of 10 mg/ml
chloramphenicol stock.
For 1 liter media, add 1.5 ml of 10 mg/ml chloramphenicol stock.
Deionized formamide: Stir formamide (Schwarz/MannBiotech 800686) with Amberlite MB resin, 10 g. per 100 ml, for one hour to deionize; filter through Whatman 3MM paper, store in a
dark bottle at room temperature or 4deg.C.
10X denaturing buffer: 200 mM Tris-HCl, pH 9.5, 1 mM EDTA, 10 mM spermidine in doubledistilled water.
2 ml 1 M Tris-HCl, pH 9.5
20 ul 0.5 M EDTA, pH 8.0
1 ml 100 mM spermidine
ddH2O to 10 ml (aliquot and
store at -20degC)
Diatomaceous earth (100 mg/ml): Suspend 10 g of diatomaceous earth (Sigma D-5384) in 100 ml of distilled water in
a 100 ml graduated cylinder, and let it settle down for 3 hours. Decant the supernatant, and resuspend the pellet in 100 ml of 6 M
guanidine HCl, pH 6.4, containing 50 mM Tris-HCl, 20 mM EDTA.
Commonly used solutions (1)--- 1 ) 2 ) 3 ) 4 ) 5 ) 6 )
Commonly used solutions (2)--- 1 ) 2 ) 3 )
Solutions for molecular cloning---1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )
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