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.
Edited by jch, 15 April 2010 - 04:47 PM.