Dr. David W. Andrews - Lab Manual

DWA LAB

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Lab Manual

biological reagents

Cell Free Systems

DNA

In Vivo Systems

misc methods

overexpression

recipes

> DNA

> CB 10X T4 DNA Ligase (+ATP)

> Plasmid Preps

> TE (Tris-EDTA Buffer)

> Buffering Phenol

> Enzyme Buffers

> DNA gel loading buffer (5 X)

> (TBE) Tris-Borate

> TBE DNA gel running buffer (Tris borate EDTA) 10 X

> Tris-Acetate (TAE)

> Transcription Recipes

Buffers

Get a printable version of this protocol (requires Adobe Acrobat)

CB5X
	RRL		WG
	volume	final	volume	final
1 M HEPES-K pH 7.5	400 uL	80 mM	400 uL	80 mM
1 M mgCl2	75 uL	15 mM
1 M MgAc2	70 uL	14 mM
0.1 M spermidine	100 uL	2 mM	100 uL	2 mM
water		425 uL		430 uL
TOTAL		1 mL		1 mL

4 NTP (5X)	UNCAPPED	CAPPED
0.1 M ATP	150 uL	150 uL
0.1 M CTP	150 uL	150 uL
0.1 M UTP	150 uL	150 uL
0.1 M GTP	150 uL	15 uL
WATER	400 uL	535 uL
TOTAL	1.0 mL	1.0 mL

adjust pH to 7.0 with 2 M Tris base

SP6 pol dilution buffer
50 mM Tris-Cl pH 7.5	250 uL of 2 M
100 mM NaCl	250 uL of 4 M
0.1 mM EDTA	2.0 uL of 0.5 M
7.0 mM DTT	70 uL of 1.0 M
50% glycerol	2.5 mL
0.1% Triton X-100	5.0 uL
Add water to total = 5.0mL

Cap (10X)L: 25 A250 units = 1.2 mg 300 æL 20 mM Tris pH 8 DTT (10X) 0.1 M tRNA 10 mg/mL

SP6 RNA polymerase:

Storage buffer

50mM Tris-HCl pH 7.9	250uL of 2M
100mM NaCl	250uL of 4M
0.1mM EDTA	2uL of 0.5M
7mM DTT	70uL of 1M
50% glycerol	2.5mL
0.1% Triton X-100	5uL make to 5mL with dH₂O