my vector size is 3.8 kb and gene size is 1191 bp. but after isolation of recombinant plasmid and doing double digestion with BamHI and NotI i am not getting proper results i.e. fallout should come around 1000 bp but it is not there.
Lane D and E contain no insert, and they are not cut at all by your enzymes. Probably your self ligation destroyed both restrict sites.
thank you Ziguo for your answer.But i really didnt understand how self ligation can destroy restrict sites. Further what can be done to avoid it.and have successful ligation.
Dear Abhijeet. Pic is already uploaded along with the question. Let me know if you need pic other than this one.
Hi Prakhar, I meant an another pic with the label which shows the band sizes. You made a nice pic but the band sizes are bit hard to interpret.
I was expecting that it might be possible that due to the higher amount of your uncut fragment the electrostatic force on the uncut fragments were more so they were pulled more and could be possible that they are appearing bit lower than what they should be. Try to use the little bit more ladder so that you can match the amount of DNA in bands.
Nevertheless, the digested fragments were not supposed to be at expected site, thus its most probably some error in the restriction digestion. I would suggest you to go for restriction digestion overnight at required temperature, vortex well the restriction buffer and enzyme well prior use, and if still error persists, go for subsequent single digestions.
will it be okay to double digest PCR product and vector overnight with BamHI and NotI. For many labs prefer 4hrs is optimum for digestion PCR product and vector.
Restriction digestion enzymes appears to be working fine. Did you check the pcr amplified gene fragment on gel and shift in vector size after ligation?
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