Hi Researchers,
Let me tell you that I was trying to isolate a gene of interest (700 bp) from a Cosmid as a template (36 kbp) and I got a PCR product of 1000 bp instead of my gene of interest! even during a temperature gradient with the primers, I got a strong band at 1000 bp approximately and also a smeared band at 700 bp (my gene of interest), and every time I tried to perform this PCR I got the same result (ADDED FILE 1.jpg).
The funny thing is that all of that was before this whole quarantine thing, and when I came back to the lab I made the same thing (performed a temperature gradient with my primers) and got a strong band at 700 bp, here, I changed of reagents (could be possible that contamination could drive such behave on amplification to get that difference on the size of the PCR products?), when before quarantine I was getting 1000 bp when I didn't even was looking for 1000 bp (ADDED FILE 2.jpg)
Also, wanted to tell you that my forward primer has an alignment 300 bp after my gene of interest (so 300 + 700 bp = 1000 bp), and this is exactly what I wanted to corroborate with sequencing because I was getting my 1000 bp band. But apparently, when I was re-amplifying that sample (1000 bp) I GOT NOTHING! (ADDED FILE 3.jpg), and if you look closer I got now a smeared band at 700 bp, not like before that I was getting a strong band at 1000 bp. So it looks like right now I can't corroborate that behave with my primers cause the PCR product is not amplifying even after changing the PCR cycle, during extension to polymerase 1000 bp. Right now looks like that amplicon of 1000 bp is a ghost...
I'm anxious to read all your answers and suggestions about this situation!
If your amplimer has a high GC region that can anneal to another high GC region creating a loop of 300 bases then the Taq may read across the neck of the loop to give a shortened amplimer. It might only take a slight variation in cycling conditions or salt concentration in the reaction to allow this. It would be interesting to see what the amplimer is if you added something that removes secondary structure in the template and amplimer. I would try either amplifying in presence of 1molar betaine or adding DMSO at between 1% and 6% final concentration in the pcr mixture to see if the 1000 base product is generated. You can use both betaine and dmso in the same reaction if the results look promising
I wonder if it makes any difference to the amplification if the cosmid is coiled or linearised?
I agree with Paul. You can try linearizing the cosmid with RE digestion that cut the template outside the target amplification region. Subsequently you can try amplifying the region of interest.
SD
Sibnarayan Datta Thank you for your answer and I actually tried it before, linearized the Cosmid with a RE making one cut in the whole cosmid to linearize it, and subsequently performed a PCR but it didn't amplify anything, maybe I can try it again and see what happens!
Paul Rutland Hi! Thank you for your answer, and it's a perfect one actually because I also could think about a loop during the PCR process but all of my PCRs have included DMSO at 5% since the beginning for the same reason (my template has high GC region), would make any difference if I use betaine? or use my DMSO at higher concentrations?
Would be interesting to see what the 1000 bp amplicon is with sequencing! but I'm not able to amplify it again because for some reason now it doesn't give me that result as before.
5% DMSO sounds good David Carmona-Berrío ...you cannot go much above 8% without inactivating the taq. If the GC is quite a long region then betaine may still work because it nullifies hydrogen bonding so everything will melt easier than usual. Any grade of betaine will do. If you use high GC buffers like promega's Q solution be careful as some of them already have both dmso and betaine so your dmso could get a bit too concentrated
Phuc Hua Hi! Of course, I'll show the thermal program I set up.
95 ºC, 3:00 (Initial denaturing time)
* 95 ºC, 0:30 (denaturing during each cycle)
* 64,5 ºC, 0:30 (annealing time)
* 72 ºC, 0:42 (extension time)
X35
*72 ºC, 5:00 (final extension time)
I think it's a great idea to denature the Cosmid during more time at each cycle it may eliminate the coiled structures it can make and interference with the isolation of my gene through the PCR. But the elongation time is actually the perfect one, what do you think?
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