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Insert phosphorylation upon Klenow?


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5 replies to this topic

#1 M!ke

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Posted 06 April 2009 - 05:55 AM

Hey guys,

I am desperately trying to clone a PCR fragment into a vector via blunt-end ligation. So far, this has not worked at all, so I hope to get some input from you how to proceed.

Here is what I do to the vector:

- BamHI/NheI double digest (fragment about 4 kb in size)
- Preparative agarose gel => cut out desired band
- QIAGEN gel extraction
- Mung-Bean nuclease: remove cohesive overhangs
- QIAGEN PCR purification kit
- Dephosphorylate
- QIAGEN PCR purification kit

In parallel, I prepare the insert as follows:

- PCR (fragment about 100 bp in size)
- Preparative agarose gel => cut out desired band
- QIAGEN gel extraction
- Klenow Fragment (to remove 3'-A added by polymerase)
- QIAGEN PCR purification kit

With this setup, I don't yield any colonies upon ligation and transformation. I have tried various vector:insert ratios, ligation times and temperatures, addition of PEG, but nothing has helped so far.

It was only today that one possible explanation came to me: Is it possible that my insert does not contain terminal phosphates which are required for the ligation? It is the first time that I am cloning a PCR product without any restriction sites, so that is why I haven't thought about T4-PKN'ing the insert yet. But: As I remove the 3'-A, do you think the Klenow fragment will leave a terminal phosphate?

I am looking forward to your replies, many thanks in advance!

Best,
M!ke

#2 phage434

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Posted 06 April 2009 - 10:56 AM

This is a protocol asking for trouble. Why aren't you doing a TA or Topo-TA cloning reaction? It's far easier, has many fewer moving parts, and will likely work the first time. If you do want to continue down this road, I'd immediately forget mung bean nuclease and switch to Epicentre End-It for blunting the insert. And why would you use a different technique on the insert and vector? A blunt ligation is also more difficult, and unnecessary in this case.

#3 M!ke

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Posted 07 April 2009 - 01:37 AM

Heya,

thank you very much for your reply! I appreciate your comments, let me explain how I ended up with this strategy:

Why aren't you doing a TA or Topo-TA cloning reaction?

This ligation is intended to insert a customized MCS (obtained by DNA synthesis) into a particular vector which is not capable of TA cloning. I must admit it is very intriguing to try Topo-cloning, but then I would still have the insert in the wrong vector, which would require yet another cloning step.

I'd immediately forget mung bean nuclease and switch to Epicentre End-It for blunting the insert.

I haven't heard about this enzyme (mix?) yet, but it does sound good... I will discuss this with my colleagues, maybe there is someone who can give me an aliquot to try.

And why would you use a different technique on the insert and vector?

That is because the insert carries 3'-overhangs, which can be cleaved off by Klenow, whereas the vector contains 5'-overhangs to be removed by Mung-Bean nuclease. I just wanted to get rid of all overhangs instead of filling some of them up, so that's why I chose this strategy.

By the way, I am pretty sure that phosphorylation of my insert will help a lot. Indeed, 5'-phosphorylation seems to be required for blunt-end ligation, and as my primers do not carry such a phosphate, this is very likely to cause trouble. I will try T4 PNK today, we will see whether I get colonies or not :)

Any further remarks will be greatly appreciated!

Thanks,

best,
M!ke

#4 M!ke

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Posted 14 May 2009 - 05:12 AM

Just in case this might be useful for someone...

The ligation finally worked after some simplification of the protocol:

1. Use Pfu instead of Taq polymerase (proof-reading => no 3'-A overhang => no Klenow+purification necessary)
2. Omit dephosphorylation of the vector (increases colony no. due to re-ligation, but again reduces the number of steps)

In the end, 2 out of 10 picked colonies showed the expected band pattern in a control digest, one of which was found to carry the perfect sequence...

As always in cloning: One single colony can be enough if it carries the correct construct... but it might take some time until you arrive there :D

Best,
M!ke

Edited by M!ke, 14 May 2009 - 05:12 AM.


#5 ddddyyyy

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Posted 14 May 2009 - 08:41 PM

I agree with M!ke










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#6 T C

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Posted 14 May 2009 - 09:22 PM

I too agree with M!ke,

I routinely use pfu to get the blunt PCR, then I phosphorylate it (if my oligos are not phosphorylated) and prepare my insert.

As a vector I use Eco RV digested pBS or Sna BI digested Litmus28 or Stu I digested Litmus28, Which I CIP (unlike M!ke) and use for ligation.

I do this routinely and get the desired clones.

Hope it helps.

Best,
TC




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