I am trying to blunt clone a 1 kb PCR fragment into pBluescript. The PCR product has restriction sites at the ends, but I have never had much luck trying to cut ends of PCR products and clone directly, so I usually clone blunt into Bluescript and then cut the band out so I know both ends are correctly cut. When I transform E. coli with pBLuescript I get lots of colonies, few when I EcoRV +SAP cut, few when I cut and SAP and ligate. Those are controls and expected results. In a parallel reaction, I ligate the RV + SAP plasmid to the PCR band purified from a gel and get lots of colonies, but they are nearly all blue (>95%). I thought perhaps I had gotten some plasmid in my gel purified PCR band since I ran some other stuff on the gel, but I got the same result when I took the PCR reaction and used a Qiaquick PCR cleanup column to purify the PCR product. I am mystified at what I could be adding from a PCR reaction that would hugely increase the number of blue colonies in a blunt ligation. Any thoughts welcome. Thanks- Dave
I would try transforming some of your PCR product to be certain the background is not coming from there. It does sound like you have some plasmid contamination from somewhere if you have tried the vector controls and they show little background.
I presume you are phosphorylating your primers or your PCR product if you have SAP treated the vector?
I would try transforming some of your PCR product to be certain the background is not coming from there. It does sound like you have some plasmid contamination from somewhere if you have tried the vector controls and they show little background.
I presume you are phosphorylating your primers or your PCR product if you have SAP treated the vector?
Hi Michael- Your answer is helpful and shows I am more out of practice that I thought. I was thinking the primers were phosphorylated, but realize now I did not specify that when I ordered them so will need to kinase the PCR product. I will also do the transformation with the PCR product alone..
Glad that helped. On a side note, I have usually had much better luck digesting my PCR product than blunt ending it. Normally if you add about 4 nt spacer at the ends, the digestion will work fine. But your mileage may vary.
If the controls gave expected results so we are sure that there is no problem with the restriction enzyme, E. coli, ligation solution, and pBluescript vector. Your techniques (the protocol you followed) are also correct. Then, my suspicion goes to your PCR fragment (the insert) or plasmid.
Personally, I would always add a few nucleotides before the restriction sites in the oligos and diggest and clone directly the PCR product. Normally I prepare a 12 uL digestion (10 uL of quantified PCR product, 1.2 uL of restriction enzyme buffer and 0.4 uL of each restriction enzyme) I incubate for 2-3 hours, and the I freeze it at -80 and melt it a couple of times. I use 4 uL of that digestion product directly as insert in a 10 uL ligation (3 hours at 20 degrees) and normally works quite well. And times are flexible!
Thanks for all the feedback. I did add extra nucleotides to the primers so the PCR product should be cuttable. I realized that the two enzymes are incompatible in buffers, so I am redoing with a sequential cut of both vector and PCR product. We shall see. Javier- I don't understand your protocol. One of my enzymes is not heat inactivatable, so I will gel isolate the double cut vector. But I don't understand your -80 routine. What is that supposed to do?
Well, one strategy that also works is to use a TOPO TA cloning kit. Eliminates the need to add restriction sites to primers and you simply add your freshly-made PCR product to the plasmid. No phosphatase treatments needed. They are a bit pricey, but work really well. I've even used them with undergraduates in a lab class.
https://www.thermofisher.com/order/catalog/product/K457502#/K457502
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