Hei everyone,
We are starting a new project and realized we have quite a lot of restriction enzymes left from a previous one. We are planning to do two double digestions and then ligate the products. One of the double digestions is performed with SpeI and EcoRI and we have quite a lot of EcoRI left but will need to order new SpeI and for practical reasons we'd like to get it from a different company. Until now the double digestion (with SpeI and EcoRI) has worked fine in the following buffer (Buffer 1):
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 at 25°C
We'd now like to get SpeI which is recommended to be used in the following buffer (Buffer 2):
50 mM Tris-HCl
10 mM MgCl2
100 mM NaCl
1mM DTE
pH 7.5 at 37°C
I would assume that the enzymes should still be active, even if you use SpeI form company 1 in the buffer that is recommended for SpeI from company 2... Any advice on whether we might be fine using Buffer 1 or 2 for both? We would greatly appreciate any help or tips!
Hi there,
I would suggest sequential digests in order to avoid any problem as buffers are quite different (especially concerning ionic strength).
Hi Sigrun,
just go through the attachment it will be very useful for your work
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