Recently I have done a cloning for sequencing of my gene of interest. I used TA cloning method and followed by transformation. After screening and selection; I extracted plasmid DNA and confirmed by agarose gel electrophoresis. I got the same size of pcr product while doing pcr for isolated plasmid DNA. But, Sequencing results say that gene of interest is missing? Could somebody suggest me how to solve this problem and where I have gone wrong?
You can use following methods to check gene of interest.
1. Plasmid DNA rapid screening test
Thirty micro liter aliquots of rapid screen lysis buffer were pre warmed at 37°C and selected colonies from grid plates were aseptically transferred into lysis buffer using sterile tooth picks. The tubes were incubated at 45°C for 5 minutes in a water bath and were placed on ice for 5 minutes. The tubes were centrifuged at 14,000 rpm for 1 minute and 15µl of supernatant from each tube was electrophoresed on 0.8% agarose gel in TBE buffer for the observation of plasmid DNA. The recombinants were distinguished by the increment of plasmid size according to the length of insert.
2. Colony PCR Confirmation
Several white colonies and one blue colony were selected from the grid plate and separately streaked on LB-Agar/Ampicilin/IPTG/X-Gal plates. Same colonies in the grid plate were touched with a sterile tooth pick and swirled in a 15 μl of PBS buffer. A 1 μl volume of the PBS buffer solution with inoculated white colony was taken as the template for the colony PCR reaction and as the negative control 1 μl of the PBS buffer solution with inoculated Blue colony was used. The optimized colony PCR assay for the gene of interest was performed with selected white and blue colonies.
one more test using restriction endonucleases to make sure the righr size
Hi there,
What set of primers did you use for the PCR reaction supposed to tell you the insert is there? Did you run the PCR reaction control (empty plasmid as a template)? To get sure of the actual orientation/localization in the plasmid you should also run a PCR with one primer specific of the plasmid backbone and one primer specific of the insert. And finally is your selected plasmid empty or did you catch something else than the expected gene?
Thank you Janith Wanigasekara. I will do the protocol and check my colonies with this method.
@ Dominique Liger
I got a single band size of interest. But, Ididn't check with plsmid backbone. I just have done only with the colony itself. I will re-do with plasmid backbone and gene of interest.
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