I have done many experiments and always i get colonies on LB-agar plates (Selection is Kana 50, Genta 25), But colony PCR always appears negataive with very good bands in positive control.
Factors to consider:
1) Is your construct resistant to both genta and kana?
2) Is the kana plates prepared fresh? If the kana plate is problematic, then both the positive control and negative colonies will grow. So this probably means that the cloning failed.
2) Self ligation - how do you prepare the vector for the cloning?
One of the reasons could be the self-ligation of the plasmid that you are using for cloning your gene. It can be avoided by using 2 restriction enzymes and gel purification of the digested plasmids and ligation with the digested PCR product
Is it possible that non recombinant plasmids causing the problem?
ligation of your gene with the plasmid may be not proper and only the plasmid containing the selection marker is getting transformed?
Rajesh Dev Sarkar Latif ur Rehman Wong Ling Chie
I did Gateway cloning (BP and LR), First BP reaction to insert my gene into entry vector (pDONR221), then did colony PCR to confirm, it was good with appropriate product length. then did LR reaction to insert the gene from pDONR to destination vector (pEG101), the did colony PCR and extracted plasmid from positive colonies using full length primers, product was great at this step, then i did electroporation and transformation of that same plasmid (insert+pEG101 from LR-DH5alpha) but there i get colonies around 30-40 per plate with no positve PCR
Swapna Priya Rajarapu Yes i did, i carried out restriction digestion to confirm product and cloning at every stage just to avoid any false positive results.
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