I'm tring to insert a fragment with 342pb in the pENTR/SD/D TOPO cloning vector, that have about 346pb. I have already purified the gel with the DNA fragment and did the ligation, using the protocol 5 minutes and 16ºC overnight. The ligation occurs, but when I make a pcr to confirm it appears lots of non specifc bands. What could I do to improve the reaction? Thanks
What primers are you using to verify? pENTER/SD/D TOPO contains fairly long inverted repeats - inverted repeat regions can wreak havoc on PCRs, especially if the primers are too close to or within the repetitive regions. Having strange bands from amplifying repetitive regions is very common.
Also, as I understand it, you're using the ligation reaction as a template for PCR, right? If that's the case, I wouldn't even bother with that, personally. If it worked at all and your transformation efficiency is decent, you'll get colonies and you can screen those.
What primers are you using to verify? pENTER/SD/D TOPO contains fairly long inverted repeats - inverted repeat regions can wreak havoc on PCRs, especially if the primers are too close to or within the repetitive regions. Having strange bands from amplifying repetitive regions is very common.
Also, as I understand it, you're using the ligation reaction as a template for PCR, right? If that's the case, I wouldn't even bother with that, personally. If it worked at all and your transformation efficiency is decent, you'll get colonies and you can screen those.
Use M13 primers rather than gene specific primers to check for the intesrt. Typically PCR with M13 primers on an empty pENTR should give you a PCR product of about 280bp so in your case you should expect a product of about 700bp. The M13 PCR is very clean and it is used for checking successful ligation in pENTR vector.
Good luck
While doing with the pcr cycles, increase the annealing temp. and try again. Generally its Ok to get two bands in ligation reaction due to the presence the vector. it also happened with me few times, but I carried it further the next step it proved gud to me.
All the best...
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