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TA cloning problems


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#1 lj269

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Posted 19 October 2004 - 04:28 AM

Hi.

I was hoping that I could get some advice on an annoying problem I'm having with some TA cloning.

I've been trying to clone 5 DNA fragments (500 - 1500bp) into the topo 2.1 TA cloning vector. Transformation into our own DH5a cells seemed to work fine with 100s of white colonies. However, after colony PCR screening using the M13 Fwd and Rev primers, none of the seven I tried for each revealed positive results. Instead, in the ones that did show amplification, there was a band about 1.9kb. This occured in colonies from all 5 inserts. PCR using my insert primers indicated 100% success but keeping in mind these could be false positives I decided to grow up and miniprep 2 samples from each (10ml overnight). PCR against the miniprepped plasmid using M13Rev and either my forward or reverse primers again suggested the insert was there but digest with EcorI (sites flank cloning site in 2.1 vector, 1ul plasmid + 2ul enzyme overnight) revealed nothing. It was hard to tell if the enzyme was even cutting. Sequencing from a different vector where I was having similar problems suggested contamination that could have been a result of overloading the Qiagen spin minprep column. Maybe that is my problem here too. Anyway, some advice or suggestions as to where to go next would be gratefully recieved. Do people think that my insert is there and, if so, what could be the problems with the digest? Could my plasmid by contaminated and could this affect my digests? Also, do people often see amplification of bands apparently unrelated to insert size when using the M13 fwd and rev seq primers?

The fact that I get positve results when PCRing using one vector and one insert primer suggests that the insert is in but the enzymes not cutting is puzzling me. I will send 2 to be sequenced to be sure but I don't know what to do in the situation that my insert is apparently in but my enzymes do not cut. Not much use for the next step!

Any ideas/tips/pointers/suggestions would be greatfully recieved.

Regards,
lj

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Posted 19 October 2004 - 05:35 PM

Hi,

Here are my two cents.

1. Did you use fresh PCR products for ligation? Did you get clear PCR bands? How did you purify your PCR products?
2. PCR screening of colonies is prone to contamination, always include a negative control
3. To verify the insert, use restriction disgestion in stead of PCR.
4. Did you load all 10 ml culture to a qiagen spin column? that is too much, you can use 1-2 ml.


Hope that helps.

#3 lj269

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Posted 20 October 2004 - 04:48 AM

Hi, thanks for the reply. I think I've realised what's wrong though, silly mistake on my bahalf. I am using a plasmid PCR template which is kanamycin resistant, however I had mistakenly thought it was amp. I didn't gel purify my PCR product and then selected for +ve TA transformant on kanamycin plates! The template plasmid is of similar size and origin to the 2.1 topo vector so I believe the M13Rev primer binds which is what was giving my postive results with the PCR screening, however it lacks any of the restriction sites (luckily) so I didn't get any digest. Just realiased all this last night. Repeated the TA cloning last night after gel purifying and plates seem more realistic (was 1000's before). In process of screening so fingers crossed.

On the subject of using 10ml o/n cultures for minipreps. I have always done this before with no problems, ofcourse I spin down and resuspend in 250ul of P1 buffer. Is this too much for the column to take? If so, do 1, 2 or 5ml cultures significantly reduce plasmid yeild or is it a matter of say 2ml being optimal and anything more just cloggs the system up? I guess copy number affects things too, all my plasmids are pBR322 derivative so low copy number I believe. The sequencing lab suggested 8hr cultures instead of o/n so this is what I'm trying now, will maybe only use 5mls of each though...




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