Kindly some one let me know the amplicon size of this TA vector(control circular plasmid with out insert provided with kit ) from LIFE TECHNOLOGIES when amplified with M13 F` and R` primer .
I have got a PDF that claims the amplicon for this plasmid is 99 bp. but when i try doing PCR i am getting an amplicon near to 160 bp. and I tried to insert a 112 bp fragment in the linearized version of it and when i do the same PCR using M13 primers i am getting amplicon near to 280 bp.
But I am confused whether the control without insert vector if amplified should give 99 bp or 160 bp. did any one try using it please let me know asap.
Hi there,
I think the PCR size product for empty plasmid suggested from your pdf is wrong. The problem is also that there are several M13 F and R primers (look at this page: http://openwetware.org/wiki/Common_primer_sequences) and their sequence are all present in the pTZ57R plasmid ( sequence from Life Technologies site : https://tools.lifetechnologies.com/content/sfs/brochures/pTZ57R-sequence.txt). When you align primers and plasmid sequence, the minimal PCR size product would be 148bp and the maximal would be 200bp (see this page for alignment: http://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=emboss_needle-I20150611-165759-0669-83618447-es) so the result you get is coherent with these theoritical data whereas you can't get 99bp PCR product with any combination of M13 primers . As a result, your cloning is probably OK as the PCR size product is consistent with the insertion in the empty plasmid (160+112 is roughly 280). I had all the info instantly from the web so when you get into trouble don't hesitate to use the data available from the net first.
Hi there,
A digestion of the plasmid with restriction enzymes cutting only around the MCS should solve your dilemma. Your cloning seems to have worked according to your PCR results (280-160=120 which is close to the 112 bp of the insert).
Alternatively, you can either use the 280 bp as template in a nested PCR targetting the insert (using the primers you used to amplify the insert) or sequence it.
All the best.
Thank you Dominique. Ur answer really helped my problem . Actually in of the PDF they show it as 99 bp amplicon for the same vector. so i was confused. Now what u said looks fair enough.. coz the max is 200 bp and i was getting band at more than 130 bp and with insert of 112 i was getting near 280 so i think my insert sat properly. I was confused if the real amplicon is 280 or not. any ways i am gonna restrict digest the plasmid and release my insert and check, but while performing colony pcr i was confused.
Thanking you fellow researcher.
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