Hi fellow lab rats,
I am having a problem with sequencing and wouldl like for some ideas as to where I can look for the issue.
So my protocol is:
1. PCR amplify gene of interest, after purification and DNA concentration determination the products are run of a gel to check the quality.
2. The purified PCR product is then ligated into pDrive Vector
3. Transformed via heat shock into E. Coli (EZ competent cells)and then plated on a 1.5% agar plate with Carbonacillin. After allowing the plate to grow overnight at 37C the plates were transferred to a 4C incubator.
4. Clones were picked and grown in LB media + Car and allowed to grow at 37C overnichgt.
5. A mini prep was performed., DNA conc determined
DNA diulted 1:10 and sequenced.
No there is no alignemnt iwth DNA star for more than 85% of the clones picked!Andt solitions.
We performed a coloy PCR and all the clones had the right band for the palsmid