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Can one use circular plasmid that contains cDNA of interest as a template


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#1 jackster101

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Posted 24 January 2014 - 03:40 PM

for PCR. I want to clone out the insert and ligate to another vector--not cure how it was initially cloned in, so couldn't pop the insert out with a digest. I did a PCR with a circular plasmid and it didn't work! 

 

Should I linearize the plasmid first--the insert is in pRetroX-Tight-Puro--see attached vector map

 

 

Thanks in advance for your help!

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#2 DeeAnn Visk

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Posted 24 January 2014 - 04:27 PM

Hi There,

 

Did you attempt PCR using *THIS* plasmid as a circular template?  If you already have, then you obviously need to try linearizing it first.  You could probably use HindIII.  Remember to purify the digest (with the PCR clean up kit?) before attempting PCR on it.  You may want to run it on a gel after the digest to make sure that you have 2 fragments of about the right size:  ~1800 pb and ~ 4900bp plus whatever size the insert is.  While circular DNA seems to be less efficiently amplified than linear DNA, it should still work--see http://amrita.vlab.c...6&sim=321&cnt=1

 

Keep in mind that things occasionally get mislabeled in the lab, so you may not have the plasmid you think you have.  Doing a few other restriction digests and running them out on a get could be very informative.  Here is a site with the restriction map for your plasmid:  http://www.snapgene....troX-Tight-Pur/

 

Finally, you need to realize that PCR may not be extremely high fidelity, depending on the kit you use.  To avoid accidentally adding in a mutation, you will need to sequence the PCR product you get and the new plasmid into which you clone it.

 

You could also confirm that you have the correct plasmid by finding some primers that should work for sequencing and send it off.  Assuming you get something back, you could then blast it against the NCBI data base.

 

Best,

 

DeeAnn Visk



#3 phage434

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Posted 24 January 2014 - 05:18 PM

The insert is almost certainly cloned into the multiple cloning site defined in your vector description. So, BamHI and EcoRI would cut the insert out, for example.

You could also use the sequence of the MCS defined to make primers to amplify the insert.



#4 jackster101

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Posted 24 January 2014 - 05:38 PM

Thanks DeeAnn and phage! Appreciate it!

 

It might me better for me to pop the full length insert out with BamH1 and EcoRI and then do the PCR. I forgot to add that I was amplifying 2 reactions using 2 diff sets of primers with diff restriction sites (since was going to insert the full length cDNA into 2 diff vectors).

 

Ist set: Xho1-Kozak-Start codon(forward primer) and BamH1-Stop codon (rev). 

2nd set : Age1-Kozak-Start codon (forward) and Mlu1-Stop codon (rev)

 

As I write this I realize that I probably should 1st get the insert out and then do a PCR with the primers (listed above) that have the restriction sites--Right? Or that doesn't make a difference. 

 

Thanks! Look forward to replies!






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