I am using a custom vector that has been provided to our lab. It has been sequenced and I have confirmed the restriction enzyme sites are present.
I ran a digestion using HindIII and BamHI on the customer vector. I then did a ligation with our insert. After ligation, I performed an agarose gel and used to a Qiagen Gel Purification kit to purify the ligation product.
At this point I am assuming that I have a plasmid with the desired insert. To check if this is the case, I performed 3 different double digestions to search for the insert; HindIII and BamHI, HindIII and EcoR1, and HindII and Xhol. My vector has all of these sites. When I run them out on a polyacrylamide gel, my original vector without an insert does not show any cutting. I have tried increasing the amount of DNA loaded on the acrylamide gel.
Ultimately we will send it off for sequencing but I am confused why my diagnostic digestion doesn't work on the original vector
why not design one primer in the vector and the other in the insert and pcr to check the existence of insert in the vector. Primers are very cheap.
I don't know why your diagnostic digestion didn't work unless something happened to the enzymes when you prepared the samples. If you had already run a digest before the ligation you can compare that digest with your latter digest. To verify your ligation otherwise, I would double check with sequencing and/or primers like Paul Rutland suggested above.
- Agreed
- Better verifying by PCR rather than restriction digestion
- I wonder if it is technical as none of your digests work
- If salt in your vector prep is causing digestions not to work then adding more DNA which actually exacerbate this problem
- if you are using 500ng to 1ug of parent vector, try doubling the amount of Enzymes and digesting for a minimum of 3 hours
- Badges
- Science topic
- Similar topics
- Gels