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.
Hi Goutham,
I would just go ahead and do a sequencing on your short PCR product to check out the exact MCS site and your DNA sequence altogether with M13 primers. Sometimes, a vector stored in a lab or from other places might have already been mildly modified by other people without noted, such as adding some other unique sites in the MCS. With the sequencing data you will know what exact you have in hand for future work.
Best,
Frank
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