Hi,
I picked up some colonies after electroporation. And I growth those colonies in liquid medium with Erytromycin (200mg/ml). I isolated plasmid after incubation overnight. I did PCR as using two primers which is one of them is belonging to plasmid, another is belonging to my insert (300bp).
After running jel, I saw almost correct band, but I tried to PCR under the same conditions again to be sure, ı didnt see any band. Thats why ı am not sure those are right colony or not.
Have you any suggestions about this situation? How can be sure right clone?
Dear Dr Bıyıklı
The gold standard as mentioned above by Dr Raza, is to have it sequenced.
It seems good you are getting growth with Erytromycin.
I also sometimes would do a restriction enzyme digest and cut my plasmid DNA's a few
times and see if I would get the correct sizes of bands after being cut out. .
There are some good web sights where you can upload your sequence and cut it and analyze on a gel and see results all electronically on the web-sight.
https://help.benchling.com/en/articles/671010-simulate-a-digest
https://www.genscript.com/tools/restriction-enzyme-map-analysis
Article Virtual restriction analysis
Best Wishes!
Before Going for sequencing, you can try for
1. Plasmid retardation (plasmid seperation On agarose gel)
2. Restriction digestion: Insert release (using RE, that used for cloning). Restriction digestion using RE specific to insert.
If the clone is positive by above meths, then go for sequencing.
Meanwhile, you can also check for the protein expression by transforming plasmid in to BL21 cells.
Best
Thank you for your kindly replies. unfortunately ı haven't check with digestion. I will try to check protein expression before sequencing. ı hope it can be express.
Best regards,
Ayse
I agree with Sunagar and Lisandra; a restriction digest is likely to be more informative.
One explanation for the negative PCR could be that the insert is in the wrong orientation.
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running