I ran restriction digestion on my plasmid just to verify my inserts and send for sequencing. my backbone plasmid is PL6 with 9843 bp containing a 3HR (877bp) and 5HR (782). My enzymes for 3HR is EcorI and NcoI and for 5HR is SacII and AFIII. I digest my inserts in different separate tubes. For 3HR for example I get two bands if check for the size the size of my insert is correct but there is another band a bit lower than plasmid size which is almost 3kb. so the first band which is plasmid is almost 8kb the second one is almost 3kb and the last one is around 800bp (my insert size) . Do you have any experience that where the problem is? since I get three bands instead of two bands (my plasmid and insert only) I cannot verify it. I appreciate your answers
Check both your donor and destination vector sequences for all 4 enzyme restriction sites. Have you tried using universal primers to amplify across your insert to see if it is roughly the right size?
Try running an uncut control and single enzyme digests as well as your double digests. It is possible to have cryptic cut sites and using each enzyme individually can help determine which one (or ones) are giving the odd band sizes.
Can you add a picture of your gel if you still get odd results?
Robert Adolf Brinzer and Amy Klocko Thank you for your kind responses. My donor plasmid is PUC57. The plasmid and insert are in the correct sites but the only problem is a band in the 3kb part. I can't run PCR for amplification cause it gives me mutations since my insert contains AT-rich regions. So I transformed XL-1 blue chemically competent E.coli with it and everything was great. So I cloned 3HR and 5HR and after the ligation and transformation, I did miniprep and ran the gel for my construct following analytical restriction digestion, I saw three bands. Could PUC plasmid contaminate the sample? If the enzymes cut other parts than the intended site then the size of the plasmid could be reduced!? However, I will share the picture of my gel after trying single and double RE.
If your entire insert is challenging to amplify, you could check the 5' and 3' ends to verify those are in the correct vector. In brief, for the 5' end, use a F primer in the vector (and unique to that vector, not found in your pUC57) and a R in the 5' area of your gene. Use a similar strategy for the 3' end. Yes, it is possible to have 2 plasmids in 1 cell and get 2 bands (it's uncommon, but certainly can happen). What does your uncut DNA look like?
- Badges
- Science method