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sequencing problems


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#1 lenkabo

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Posted 22 March 2013 - 04:34 AM

Hi all,
we are doing our clone library on 16S rRNA gene and have some big problems with our sequences. PCR products for cloning were checked on nanodrops and gel and looked very clear with the right sizes (approx 1500bp) and single bands. We used TA cloning kit from Fermentas. Ligation did not look perfect,but was quite ok and visible on the gel. Blue white screaning were fine and positive transformants and white colonies were picked and planted separately. For the clone PCR we used M13r and M13f primers which gave us vector sequences and probably were not specific for the vector we used. We also tried to extract plasmid and PCR them with T7f and M13r primers, which gave us the product of approx. 2000bp and after purification and quantification the sequences did not work and gave us results of 150bp which was not reasonable.
We would be happy to read all your suggestion and solve our problems out.
Thank you

#2 phage434

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Posted 22 March 2013 - 01:09 PM

Is your initial sample mixed (otherwise, you could just sequence the results of the first PCR).

Does the Fermentas TA cloning vector have M13F and R primer binding regions?

When you say "Gave a product of approx. 2000 bp" do you mean it was expected to, or actually that it did? What does "quantification" of the sequences mean?

Why did you think that T7F and M13R would be good primers?

Did you use a Taq containing enzyme for your initial PCR? You must, if you are doing TA cloning.

#3 lenkabo

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Posted 22 March 2013 - 01:46 PM

Is your initial sample mixed (otherwise, you could just sequence the results of the first PCR).

Does the Fermentas TA cloning vector have M13F and R primer binding regions?

When you say "Gave a product of approx. 2000 bp" do you mean it was expected to, or actually that it did? What does "quantification" of the sequences mean?

Why did you think that T7F and M13R would be good primers?

Did you use a Taq containing enzyme for your initial PCR? You must, if you are doing TA cloning.

Well, M13 was unspecific primer (thats why we got vector sequences probably) and thats why we decided to go for T7 which is specific for the vector we used.
Before sending to the company for sequencing we just quantify our plasmid on nanodrop. We have tried many other options,combinations, checked every step and everything looked fine.
Checking our products on the gel, all sizes looked ok as we expected, so we cant really understand what is going on with our clones.
Thank you for the help:-)




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