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Problems with TOPO cloning


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#1 Agnes

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Posted 30 September 2004 - 05:31 PM

Hello,

I did a ligation for a 1.4 kb band using the pCR 2.1 topo vector. After I did the transformation (top10 cells), plated the cells, picked 10 white colonies, and cultured them over night in an LB/Amp solution.

I did a PCR check on the cultures (2.5 uL in total volume of 25 uL) using M13 forward and reverse primers. After I ran it on a gel, I got mostly 400 kb bands per sample, except for a sample with a 900 bp band. Can you tell me why I don't see my 1.4 kb insert?

I made my insert using reverse transcriptase PCR from rRNA. After I ran it on the gel I got a 400 kb band and a 1.4 kb band. So I gel purified my band. I don't understand where these small inserts are coming from... please help, thanks.

Cheers,

Agnes

#2 trinoceronte

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Posted 30 September 2004 - 06:59 PM

Dear Agnes:


The small fragments can be molecules that got stuck in the big band (1.4) and did not migrated with its proper molecular weight. If you have such a very strong band you can purify it twice from gel and then give a couple of cycles again with the polymerase and nucleotides just to enable the ends for the TOPO vector.
400 will ligate with much higher efficiency than 1.4 so you can also try to make an optimal PCR that does not produces the 400 band

Good luck !

Trinoceronte

#3 kant0008

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Posted 30 September 2004 - 08:42 PM

Hi,
the fact that you got 2 PCR products makes me think that your primers can bind twice along the template, one inside the other. That means that if you provide the 1.4 kb sequence as your template you theoretically should get 2 bands again. But the smaller one will be easier to amplify, so you might get bias. Try to do a restriction digest on your plasmid from transformed cells, with an anzyme that you know the sites for and see what size fragments you get. Good luck!

#4 kawaka

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Posted 01 October 2004 - 05:24 PM

Before you conclude you have got wrong insert, do a miniprep and digestion, which will definitely tell you the right size of your insert since PCR sometimes is hard to predict.

the small fragments can be molecules that got stuck in the big band (1.4 kb) and did not migrated with its proper molecular weight.


Interesting explanation.

Edited by kawaka, 01 October 2004 - 05:26 PM.


#5 Agnes

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Posted 01 October 2004 - 08:08 PM

Hi,

Thanks for all your helpful suggestions. I think I will plasmid prep my 900 bp band and then sequence it. There might be the possibility of having a truncated insert?!

After, I will try your ideas on the restriction digest and PCR... hopefully, it will work this time. :)

Cheers,

Agnes




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