I'm an undergrad molecular biology student and I'm trying to extract a plasmid to use as a vector. I'm going to amplify my gene of interest (from a different plasmid in a different strain) using PCR (with primers that have particular restriction sites). Then I'll do a restriction digest on my PCR product, as well as on the vector plasmid, and then attempt to ligate them together. I will attempt to transform the ligated product into electrocompetent cells.
Should I Use the Qiagen Spin Miniprep kit, or the Qiagen Plasmid Mini kit for my plasmid extraction? My lab has both kits. I'm not sure how old they are, it could be several years - do they expire with time (I couldn't find a date anywhere). Lastly should I run my product through a column after the digest but before the ligation, or again after the ligation but before the transformation; and if so, which kit should I use the columns from?
Thanks so much!
Thank you for your question. If you want to extract a plasmid, you need a kit, you could use the Qiagen Plasmid Mini kit. When you begin to use a new kit, you have to comply with the instructions.Addtional, I suggest you buy a new kit. before the transformation, you should clean your product. you can use the TaKaRa Agarose Gel DNA Purification Kit.
Thanks all for your help! I appreciate it.
Matthias R. Schaefer Is the Miniprep supposed to have a lower purity ? It seems like a less intense protocol. Regarding my lab, it is a small lab so I don't have anyone to ask right now who would know about this other than my PI
Göran Hjälm
Thanks, I did add some nucleotides upstream of the restriction site and the digest worked. I didn't think about the end fragments inhibiting the ligation but that makes sense, so I'll clean it up before the ligation. Why do the spin columns allow the end fragments to flow through though, aren't they supposed to bind DNA?Jie Cheng You suggested buying a new kit - is that to avoid potentially contaminated old kits?
If you really want to try use the PCR (or gel) column for miniprep column, the answer is that--yes it works. I did it but I do not know whether it affected the efficiency.
I used ten-year old Qiagen kit. It works but efficacy apparently lower than new one. It is workable but if your time is more precious, do not try to save money.---not worth it.
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