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Restriction Enzyme Cloning Manual Buffer Recipes



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TE Buffer (Catalog No. T9285)

(for DNA storage and standard manipulations)

10mM Tris-HCl pH 8.0 (Catalog No. 93362)
0.1mM EDTA (Catalog No. E6758)

Tip 1: DNA is quite stable in TE buffer at 4ºC. If stored in elution buffer or water then freezing at -20 ºC is advised.

Tip 2: TE can reduce some enzymatic reactions, for example, DNA sequencing reactions and occasionally ligations. Elute DNA to be ligated or sequenced in either elution buffer or water.

Tip 3: Alternatively, if large numbers of samples need to be stored, or ambient long term storage of plasmid DNA samples is required, consider Biomatrica’s DNAstable products (Catalog No. 93021-001).

Elution Buffer

10mM Tris-Cl, pH 8.5

Tip: Elution buffer is basically TE without the EDTA which is the component that can disrupt enzymatic reactions. DNA is more stable in this than in water.

Nuclease Free Water (Catalog No. W4502)

To make nuclease free water, fill a small clean lab bottle with deionised water filtered through a 0.22µm pore filter using a syringe and then autoclave. Replace every few weeks/months to avoid contamination.

Tip: Tissue culture grade nuclease free water is available from many suppliers, but you can just make it yourself. You will only be using small amounts so if you do buy it will last you a while if it doesn’t become contaminated.

10x Ligation Buffer

0.5M Tris-HCl (pH 7.6)
100mM MgCl2 (Catalog No. M8266)
100mM dithioltheritol (DTT) (Catalog No. D0632)
500μg/ml bovine serum albumin (BSA) (Catalog No. A7906)
Made up in water

Tip 1: This buffer is stored in small aliquots at -20°C.

Tip 2: The BSA can precipitate after freezing and should be re-suspended by vortexing or warming to 37°C before use.

0.5 M PIPES buffer

Dissolve 15.1 g of PIPES (Catalog No. P8203) in 70 ml of pure water

Adjust pH to 6.7 with 5 M KOH

Add water to bring volume up to 100 ml

Sterilize the buffer by passing it through a 0.22 µm syringe filter

Store at -20ºC

Inoue Transformation Buffer

Component Final concentration Amount per litre
55mM 10.88g MnCl2.4H2O
15mM 2.2g CaCl2.2H2O (Catalog No. L3306)
250mM 18.65g KCl (Catalog No. P9541)
10mM 20mL of 0.5 M PIPES pH 6.7 solution
H2O Up to 1 litre

Sterilize the buffer by passing it through a 0.22 µm syringe filter.

Store at -20°C

SOB (Catalog No. H3022)

20 g/L Tryptone (Catalog No. T2559)
5g/L Yeast extract (Catalog No. Y1625, Y1626)
0.5 g/L NaCl (Catalog No. S3014)
2.5 mM KCl (Catalog No. P9541)
10 mM MgCl2* (Catalog No. M8266)

Make up to the final volume and autoclave

*The MgCl2 must be added after autoclaving from a 1 M stock.

Materials

     
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