Double digestion problem - (Jul/25/2005 )
Hello there,
I've been trying to cut 300 bp segment from G77 vector with Nco1 and Spe1 restriction enzymes but always ending up with very poor two bands (4.7kb bigger band- I am interested in and 300 bp band, I want to get away with). Between these two bands I get a smear. I have tried using diiferent concentrations of restriction enzymes over a period of two hours to overnight but the results were same i.e poor yield. When I ran my uncut plamid along side, I noted that the yield of miniprep is very low in the the first place from this strian of bugs( E.coli HB2151, so it was obvious to expect poor yeild of dna bands upon digestion. I decided to transform DH5alpha and TOP10 (invtrogen) E. coli strains with this G77- 5 kb vector which had been prepped from HB2151. This time I got very good plasmid dna yield (but not typically looking profile of plasmid dna as one would expect on gel- got only one very-very bright band, no nick or circular dna, relaxed dna as one would expect). However, I proceeded with restriction digest with Nco1 and Spe1 but ended up with three bands. Two bands of 2.3kb and one band of -300 bp (the one I am not interested in). This was seen with minipreps from both the strain, however the yield was very high. What could be the reason for ending up with additional band? Where is the possibility of contamination? Can anyone advise me how to increase the yield of my 4.7kb vector, apart from using more plasmid dna and restriction enzymes in restriction digest mix. Looking forward to hear. Thanks
SV
Hello there,I've been trying to cut 300 bp segment from G77 vector with Nco1 and Spe1 restriction enzymes but always ending up with very poor two bands (4.7kb bigger band- I am interested in and 300 bp band, I want to get away with). Between these two bands I get a smear. I have tried using diiferent concentrations of restriction enzymes over a period of two hours to overnight but the results were same i.e poor yield. When I ran my uncut plamid along side, I noted that the yield of miniprep is very low in the the first place from this strian of bugs( E.coli HB2151, so it was obvious to expect poor yeild of dna bands upon digestion. I decided to transform DH5alpha and TOP10 (invtrogen) E. coli strains with this G77- 5 kb vector which had been prepped from HB2151. This time I got very good plasmid dna yield (but not typically looking profile of plasmid dna as one would expect on gel- got only one very-very bright band, no nick or circular dna, relaxed dna as one would expect). However, I proceeded with restriction digest with Nco1 and Spe1 but ended up with three bands. Two bands of 2.3kb and one band of -300 bp (the one I am not interested in). This was seen with minipreps from both the strain, however the yield was very high. What could be the reason for ending up with additional band? Where is the possibility of contamination? Can anyone advise me how to increase the yield of my 4.7kb vector, apart from using more plasmid dna and restriction enzymes in restriction digest mix. Looking forward to hear. Thanks
SV
There are two possibilities: (1) You have an extra RE site in your plasmid (remember not all plasmid maps are correct
), or (2) you are getting star activity (non-specific cutting) from one of your RE. You will need to do some controls using each of your RE to see which one is causing the problem. Do this test with the buffer that came with RE and also whatever buffer you were doing the double digests with.