I have a plasmid vector that I have chosen two restriction sites on, unfortunately these two are only 45bp apart and I fear that gel electrophoresis will not be accurate enough to show if my double digestion did in fact work or not. Is there an alternate method to verify my double digestions worked if I cannot rely on gel electrophoresis?
Smaller inserts are always hard to validate. Here are some options.
1. You can PCR validate the insertion by taking one primer from the vector backbone (T7 forward primers are commonly available) and one from within the insert.
2. As Paul Rutland suggested, digest the construct using one enzyme against the backbone and one against a site within the insert. The fragment sizes will indicate if there is an insert.
3. If you have unique restriction sites within the insert that are not on the backbone you can digest to see if the plasmid is getting linearized. Use the uncut vector plasmid that was used to make the clone as a negative control.
4. Sequence your construct. Will take some time to get the results but the proof is definitive.
PAGE will show you a visible band of 45 bp ( its existence shows the double cut so you do not need to see the size difference in the large fragment)
Using agarose you could do the double digest and take an aliquot which you then cut with an enzyme in the backbone of the plasmid.This third enzyme is chosen to give a fragment which includes the 45bp fragment but it should be a fragment where full size band and full size minus 45bp are well separated in agarose eg 150bp and 105 bp as the variable fragment. The existence of the 105 fragment shows that the 45bp has been cut out. Bands common to the uncut and triple cut plasmid can be ignored
Smaller inserts are always hard to validate. Here are some options.
1. You can PCR validate the insertion by taking one primer from the vector backbone (T7 forward primers are commonly available) and one from within the insert.
2. As Paul Rutland suggested, digest the construct using one enzyme against the backbone and one against a site within the insert. The fragment sizes will indicate if there is an insert.
3. If you have unique restriction sites within the insert that are not on the backbone you can digest to see if the plasmid is getting linearized. Use the uncut vector plasmid that was used to make the clone as a negative control.
4. Sequence your construct. Will take some time to get the results but the proof is definitive.
If your concern is to observe the 45bp band then run a 2% agarose gel or a polyacrylamide gel (12%). You have to run the gel for a longer period of time. If you wish to run an agarose gel instead of PAGE, use sodium borate/lithium borate buffer for electrophoresis where the voltage can be increased to speed up electrophoresis so that a gel run takes only a fraction of the usual time.
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