2 replies to this topic

[thegene]

Hi mates,
I am performing a sequential restriction for a plasmid with two enzymes that cut once.
I am using two enzymes from different companies and what i have done after restriction, i just heat inactivate the enzyme and used an amount of the first reaction to the second reaction where i added the buffer that matches the second enzyme.
Is it necessary to purify the first reaction or it works with heat inactivation since i thought about what happens to the buffer from the first reaction? would it affect the second reaction even if i heat inactivated the enzyme

Best

Edited by thegene, 01 March 2010 - 03:04 AM.

[scolix]

I usually precipitate the DNA in between the digests. After the final digest, you could heat inactivate if necessary.

[dbe]

thegene, on Mar 1 2010, 06:03 AM, said:

Hi mates,
I am performing a sequential restriction for a plasmid with two enzymes that cut once.
I am using two enzymes from different companies and what i have done after restriction, i just heat inactivate the enzyme and used an amount of the first reaction to the second reaction where i added the buffer that matches the second enzyme.
Is it necessary to purify the first reaction or it works with heat inactivation since i thought about what happens to the buffer from the first reaction? would it affect the second reaction even if i heat inactivated the enzyme

Best

Are the two enzymes not able to be in the same buffer?  Even if the manufacturer supplies one buffer, they may list a range of acceptable buffers in the product literature, and you could just make your own.

If not, I usually gel purify, which has the added benefit of selecting digested DNA - as opposed to undigested, if the reaction only cut a portion of your material.