Double digestion using EcoRI and HindIII with pHAT-CAT - (Apr/15/2010 )
Was wondering if anyone could help; i have performed a double digest using EcoRI and HindII on 6 minipreps of pHAT-CAT plasmid, eluted from the E.coli cells used to grow this plasmid. Both of these restriction enzymes were expected to give two distinct bands of known sizes. The picture of the gel does how these two bands however, all 6 minipreps have given partial digestions.. as well as the two expected bands there are 3 bands in each lane (2 lanes have 4 bands altogether).
I dont understand how this happened, both enzymes only have 1 restriction site each in the multiple cloning site. So why were more than 2 bands formed? Has anyone seen this before?
During the steps of purifying and eluting the plasmid from the E.coli cells (using Sigma GelElute Plasmid Miniprep Kit), a few mistakes did happen: 1) miniprep number 6 has less lysis solution. 2) The gilson pipette used was off so around 20% extra volume of neutralisation solution and column preparation solution were added to all of the minipreps. 3) After the plasmid had been eluted from E.coli, tube 6 (of miniprep number 6) fell and had only a little bit of solution left to transfer into the new tube.
I thought that perhaps extra neutralisation solution or column prep solution may have caused star activity in all of the minipreps, hence the partial digests in all of the lanes?
If anyone has any suggestions as to what went wrong i would be very grateful.
Three bands might be explained by partial cutting with one of the enzymes. Highest is single cut, then the two fragments at lower positions. It might be possible you have some uncut DNA as well, which might explain a fourth band as supercoiled DNA. I'd suggest that you might be trying to cut too much DNA. 1 ug in 50 ul with 5 units of enzyme for an hour would be a good place to start.
Single digests of your DNA might also help elucidate what is happening.
Thanks for your reply. It as helpful.
The additional unexpected bands which I saw were around 4000bp in all 6 minipreps. Also, two of the lanes had additional bands at arounbd 6000bp. Whilst the two expected bands (of around 3000bp and 1000bp) were found in all minipreps, could the extra bands at 4000bp be due to partial digestion by one of the enzymes and the 6000bp band be due to super-coiling as the plasmid was not cut properly?
Yes, that sounds like the situation. I suggested some ways to try to establish this. Run digestions with both enzymes individually, both together, and a lane with uncut DNA. Do the digestions as I suggested, and run 20-30 ul of the result on a gel. You should have no trouble seeing the bands, and will be able to see where uncut DNA, single cut DNA, and the cutting efficiency of each of the enzymes independently.
Thanks for the advice and suggestions!