After PCR using Q5 pol, I ligate the insert into pCR-Blunt using T4 ligase. Transformation into DH10 is successful and a few colonies are mini-prepped. The plasmid is then cut with EcoR1-HF and run on a 1% agarose gel. I should get a band for the vector at 3.5kb and a 500bp band for the insert, but I end up with no band at 3.5kb and bands at multiple lengths no where near the expected 500bp for the insert. The total protocol, for all of the samples, were conducted at the same time. Attached is the gel.
Any ideas?
Hi ,
You could have probably
1)eluted a different plasmid due to a contamination in the water ,
2) there was some kind of ligation without the 360 insert so try to change the ligation ratio to a higher ratio like to 1:5 if u used 1:3 before,
3)run the uncut plasmid in the same gel to check if EcoRI worked or not.
Well if your ladder is GeneRuler 1kb Plus then in the first three columns you do have a line at 3.5 Kb. Sample 3 is a self ligation without insert, and Sample 1 and 2 could be concatamers, constructs with multiple inserts (4 and 3, respectively). I would probably decrease the ligation time, and maybe check more colonies next time. Good luck.
I appreciate the answers. @ Purnima: I used the highest amount of DNA available and tried newly autoclaved water, to no success.
@ Nora: I will try to decrease the ligation time. Do you have an explanation for a band that shows at 300bp?
Nora,
No 300bp. I tried to do the same protocol with a different insert, 500bp, with the suggested improvements, but again got a band at an unexpected length. There is no DNA which should have ran to 300bp, but my gel showed a band at 300bp. I was just wondering if you may have known a possible culprit for the incorrect band. Thank you for your help.
Kanomi,
Sorry, I thought you meant a band on the gel you attached. It is indeed very strange to get a shorter fragment than any of the components after a ligation. I would really suspect contamination in this case.
Nora,
Just as feared and suspected. Thank you for your assistance. I appreciate your time.
Kanomi
No worries Kanomi, and don't despair: the more trouble-shooting you are forced to do with a technique, the better you get at it!
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