I am trying to select E.Coli colony which has inserted gene on pk18mob plasmid from E.Coli DH5 alpha transformation. After transformation, I did colony PCR reactions from some small colonies and it showed my inserted gene on gel electrophoresis image.
However when i cultured these colonies in LB liquid culture and extracted plasmid for restriction enzyme reaction, it did not show my inserted gene on gel electrophoresis image.
My RE protocol is 1ug of plasmid, 1ul BamHI, 1ul HindIII, 5ul buffer and water to 50ul at 37oC for 15 minutes. Then pipet 10ul to gel electrophoresis.
Tung Anh Le
Can you share with us the gel photograph after restriction digestion and colony PCR also?
Also please describe the length of your insert size.
Thanks
were your primers both inside the insert sequence or one in the plasmid and one in the insert?
If both were insert primers then you may just have a positive pcr from wetting of the plate with ligation mix and insertionn may not have taken place at all
Paul Rutland after transformation i select small colonies and streaked these to another LB plates then took pcr test. I also tested my primer with plasmid DNA using PCR as positive control and it did not show any bands on gel imgae.
Did you maintain selection on the streak plates & broth culture? If not, your bacteria can drop the plasmid.
I am assuming you grew an overnight broth culture to use for plasmid DNA extraction and then PCR. If you tried using the same tip to streak the new plate for colony PCR, then what you are seeing is just plasmid from your ligation.
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