I am pretty new to molecular biology techniques and just when I think I have it all figured out, something stumps me. This is how this gel (1.5% agarose) is loaded:
1. GeneRuler ready to use cat# SM0333
2. empty
3. pGEX-2T digested with BamHI
4. pGEX-2T digested with EcoRI
5. Undigested pGEX-2T isolated from BL21(DE3) using Qiagen miniprep kit
6. Digested PCR product
7. Digested PCR product
8. pGEX-2T digested with BamHI and EcoRI
Gel stained with SYBR Safe and imaged using blue channel on LiCOR odyssey.
I am confused as to why the linear DNA in lanes 3 and 4 is running smaller than the undigested DNA. I'm also confused as to exactly what the two bands are in lane 5? Please help.
Based on electrophoretic mobility, from fastest moving in agarose gels to slowest, plasmid DNA can be Supercoiled denatured DNA > Supercoiled > Linear DNA > Relaxed circular DNA > Nicked open-circular. Since Lane 3 and 4 should ideally be linear DNA cut using a single restriction enzyme, Lane 5 must contain Nicked open-circular DNA and Relaxed circular DNA.
Nicked DNA will have one or more strands of the plasmid DNA cut but the circular profile remains intact. So all superhelical tension will be lost and the plasmsid will look like a big floppy circle. Relaxed circular DNA consists of plasmid DNA that has been nicked and patched back up. This form should present as multiple bands or a small gradient of DNA bands. Depending on the extend of the nick, base stacking interactions and hydrogen bonds can take the place of the missing phosphodiester linkage and retain the structure of the DNA. So there will be loss of some tension in the superhelix but it remains to be somewhat intact, but floppy, and migrates slower than the linear DNA.
I happened to read a PNAS paper in order to get some background on DNA nicks (doi:10.1073/pnas.87.7.2526). I found this really cool, so if you or any one else is interested about how DNA nicks are stabilized by hydrogen bonds, this paper should serve as a primer.
Finally, good quality plasmid extracted from the bacteria should majorly be supercoiled in conformation. But occasionally, I too exclusievly get these slow moving forms. I think, this happens when DNA gets sheared in solution during the alkaline lysis protocol. I usually mix the solutions by inversting them gently and avoid pipetting the sample too much. Also, overdrying the DNA during alcohol preciptation or letting the mini prep column dry out before elution can also break the phosphodiester bond in DNA and result in nicks.
I am not an expert on this subject, but I did enjoy researching a solution to this problem. I hope someone else can corroborate and add more to what I said.
Hope this helps.
Praveesh Valissery Thank you. I was mostly confused as to why I saw no supercoiled DNA in lane 5, but you answer makes sense. Thank you again!
Hi:
I think the BL21 is not a good E.coli for plasmid, but for protein. it is EndA+. it might be the reason that you did not see supercoiled DNA. see the link:
https://www.researchgate.net/post/Plasmid_prep_from_BL21_cells
Junjie Shao thank you. I did not have a DH5a stock of bacteria containing this plasmid, so I needed to isolate from the stock of BL21 for expressing protein.
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