I inserted 3131 bp to a plasmid with 3600 length by ECORI and BSP119I enzymes
After transformation of bacteria (TOP10) and replication, I purified the plasmid.
Plasmid digestion and sequencing showed that only 804 bp from 3131 bp was cloned.
I repeat transformation with other strain of Ecoli (BL-21) but I got the same results.
Sequencing is showed that cloning carried out at GAATTT site (804 bp) instead of GAATTC (ECORI site) at the end of 3131 bp fragment.
We were cut 3131 bp from gel after digestion with ECORI and were extracted the band from gel. therefore we removed the ECORI and not existed at ligation reaction for creation of star activity.
Can you help me?
Inclusion of agarose gel photographs with appropriate DNA fragment size markers may be helpful to resolve your intriguing observation. Although it is possible that the E1B may be expressed in bacterial cells, it is unlikely. Other possible explanations are likely.
So, looking at the GAATTT problem, did you send the plasmid or gene for sequencing?
If gene, could it be because the there is an error (causing mutation while amplification). the answer might be amplify again and try transforming.
If plasmid, might use another batch of plasmid because at some point, a mutation might occur.
Also, a 3131bp gene is very large, so inserting into a small plasmid is quite hard, and, using a high quality Taq polymerase could help because they have long read lengths whereas normal quality taq only accurately reads about 700-1000 bps. If the Taq polymerase reads for too long, the front and the end regions of the gene might cause an error.
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