I have a linear PCR Amplicon of 8715 bp. It has BamHI sites at its ends with 5' flankers of 15 bp at each end. I need to digest it and clone it. However, I have a feeling that it is not being digested properly. Is there a way to ensure digestion? Also, will the digested product (8680 bp) be visible on the gel, after digestion, at a position lower than the undigested amplicon, if run on an agarose gel?
Help is appreciated.
The enzyme works fine. Its tried and tested with pDNA. Whether my PCR amplicon is getting digested or not, that is my key interest. My personal experience says that not all flankers augment/allow digestion properly. If the digested product would be visible at a lower coordinate on the gel, it would have made things easy.
You can amplify your digested fragment with primer beginning in the flankers region and with only 3-4 bp in the intern 8680 bp region. If you do not get PCR fradments, was the digestion successfully. As control amplfy also the intern fragmant.
Hey Rohit- I would recommend gel purifying your PCR product and then ligate it into a simple vector (with no MCS) I have used #3271 T-Vector pMD19 from Takara and had success. I would the use a product like #6024 TaKaRa DNA Ligation Kit LONG for your ligation reagents. Ligate overnight at 16 C 10uL. Then transform all into 100uL competent cells (DH5alpha). Be very gentle after the 42C heat shock and let the tubes sit on ice 3-5 min. Let cells recover in SOC ~1 hour at 37 C shaking, spin down the RX, decant most of the media & plate all of the cells. The next day you can grow-up ~ 4mL cultures of some colonies and then plasmid purify. NOW ! you can cut your band out with confidence that it is being cut & proceed with your further cloning ... I know that this adds a few extra days/steps, but in the end, it should work out for the best!
happy cloning-
mike
Hi Rohit,
sometimes it could be useful to use the high fidelity version of the restriction enzyme, when the condition are not optimal. I always use the high fidelity one to digest PCR product.
Good luck!
Valeria
Yes, gel electrophoresis -- remember to run the optimal % gel for your fragment size & also run undigested sample. NOTE: If your starting material is circularized it will run much smaller than if it is linear, so you would have to take that into account. Alternatively you could use a single cutter and liniarize it.
Best-
mike
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