Re: Re: Re: Trouble about double digestion ok but auto-sequencing says no signal
Original Message1) is it possible that the cloned plasmid passes double digestion test but failed in sequencing?
Thanks a lot for your reply, BlueDevil. I used vector primer, RV3 to sequence the plasmid, and at the same time the other clone had been successfully sequenced. About the plasmid quality problem I guess I would rule out by the second time sequencing because that I sent them the streak strain. If there's any wrong conclusion please correct me. I wonder two things: 1) is it possible that the cloned plasmid passes double digestion test but failed in sequencing? what reasons cause it? 2) will re-transform the plasmid and send the new colony afterward solve the problem?
Thanks a lot.
With molecular biology, anything is possible. I'm assuming you are using a selectable marker, e.g. ampicillin resistence. Is that correct? If so, and if we assume the selection agent is at the correct concentration and the plates are not too old, the bacteria must contain plasmid. The fact that you carried out double digestion successfully also rules out the absence of plasmid. Although seeing a band of the expected size (was it?) on a gel after double digestion is very suggestive of the presence of your insert, only sequencing will tell you for sure. If another clone in the same vector was sequenced successfully, it sounds like something is wrong with your plasmid prep. You might try re-isolating your plasmid, and the plasmid containing the insert that was successfully sequenced, and sending them in at the same time. If neither sequence, it's the prep. If only the one that worked previously sequences, then it's the new clone.2) will re-transform the plasmid and send the new colony afterward solve the problem?
It might, but if the problem is in the prep, it probably won't. How are you purifying the plasmid? Even with a good plasmid purification kit, and depending on the bacterial strain, it sometimes takes some additional clean-up to get things to work. I would suggest an extra ethanol precipitation; make sure you wash the pellet thoroughly with 70-75% ethanol before air drying. Resuspend the plasmid in water and make sure your water is very good. The same goes for the primers. Did you include primers for sequencing from both directions?