19 replies to this topic

[jiajia1987]

scolix, on Feb 6 2009, 12:50 AM, said:

jiajia1987, on Feb 4 2009, 04:29 PM, said:

scolix, on Feb 4 2009, 08:32 PM, said:

I would leave it for an hour. It should be plenty of time.

You could try site directed mutagenesis instead of error prone PCR.

Hello,

Thanks for your reply. Would it be better to do a double digestion at the same time, or is it usually better to do a sequential digestion with PCR cleanup in between?

If you are using compatible buffers, then I would do a double digest. Saves time.

I am using BamH1 and Nde1 on my PCR products while BamH1, Nde1 and Nco1 on my vector. They do not use the same buffers, but the buffer recommended for the combination would be NE Buffer 3 or BamH1 Buffer. Would you still do a double digestion?

[arush]

yes but u'l loose both sites in the process!!! (this is in response to the original question)

Edited by arush, 06 February 2009 - 06:23 AM.

[jiajia1987]

arush, on Feb 6 2009, 11:22 PM, said:

yes but u'l loose both sites in the process!!! (this is in response to the original question)

Hi Arush,

which original question do you mean? Are you referring to the below?

"Hi everyone, I just wanna ask how long do you guys usually leave your enzyme digestion? Is 3 hours enough?
Has anyone tried making mutated libraries using error-prone PCR and doing an enzyme digestion on the PCR products so they can be cloned into vectors?

Thanks in advance. "

If yes, i did think that error-prone PCR will cause some mutations in the restriction sites. However, the mutation rate will not be so high that the restriction sites will be lost on every single copy. The mutation is also random. So, I will still get back some copies that are mutated somewhere else other than the restriction sites. Right?

[scolix]

jiajia1987, on Feb 6 2009, 03:04 PM, said:

I am using BamH1 and Nde1 on my PCR products while BamH1, Nde1 and Nco1 on my vector. They do not use the same buffers, but the buffer recommended for the combination would be NE Buffer 3 or BamH1 Buffer. Would you still do a double digestion?

According to NEB buffers,
BamHI -
NEBuffer 2:             100%
NEBuffer 3: 100%
NEBuffer 4: 100%

NdeI
NEBuffer 2: 100%
NEBuffer 3: 75%
NEBuffer 4: 100%

NcoI
NEBuffer 1: 100%
NEBuffer 2: 100%
NEBuffer 3: 100%
NEBuffer 4: 100%

As  you can see, one could do a combined digest with either buffer 2 or 4. I might even do with buffer 3 as the enzyme would work 75%.

I agree BamHI comes with a special buffer but it definitely works with the other buffers equally well.

[jiajia1987]

scolix, on Feb 7 2009, 03:54 AM, said:

jiajia1987, on Feb 6 2009, 03:04 PM, said:

I am using BamH1 and Nde1 on my PCR products while BamH1, Nde1 and Nco1 on my vector. They do not use the same buffers, but the buffer recommended for the combination would be NE Buffer 3 or BamH1 Buffer. Would you still do a double digestion?

According to NEB buffers,
BamHI -
NEBuffer 2:             100%
NEBuffer 3: 100%
NEBuffer 4: 100%

NdeI
NEBuffer 2: 100%
NEBuffer 3: 75%
NEBuffer 4: 100%

NcoI
NEBuffer 1: 100%
NEBuffer 2: 100%
NEBuffer 3: 100%
NEBuffer 4: 100%

As  you can see, one could do a combined digest with either buffer 2 or 4. I might even do with buffer 3 as the enzyme would work 75%.

I agree BamHI comes with a special buffer but it definitely works with the other buffers equally well.

I am not too sure why BamH1 comes with a special buffer. Is it because of its STAR activity? Maybe that is why I get my ligation but when I run a gel to check my inserts, there are many bands? But then again, when I do a colony PCR on the cells transformed with the ligated products, I get only one band, which is the insert band of the right size. I am puzzled at why these two gives different results.

And, I checked out NEB's double digest finder, it says NEBuffer 3 is the best buffer for the three combination, though I did think of using NEBuffer 2 or 4. But my supervisor told me to use BamH1 buffer, so I don't have much of a choice.