I have a construct having Restriction site SmaI+KpnI on both sides of gene. I want to lift the gene with SmaI and KpnI. SmaI linearized it, after it i purify with PCR kit and digest with KpnI. Desired band released but very faint while very thick band of linearized plasmid. KpnI is working as confirmed by single digestion and I have also used it with NotI for lifting this cassette.
SmaI (thermo sci)= 30C
Fast dig Smai (themo sc)=37C
KpnI=37C
Thanks,
Best Wishes
If both your enzymes work as expected during a single digestion there should be no reason why the enzymes should not work when used as the second digesting enzyme. These enzymes have incompatible buffers so perhaps increase the wash steps during the pruification. What is the size of the insert compared with that of the plasmid?? If for instance you are cutting a 500 bp fragment from a 5 kb plasmid your gene fragment will be 10 times fainter than the vector simply because there's more DNA. Do you have a picture of the gel? Perhaps, if you left the gel to run for a long period it could even be something as simple as the fragment moving into part of the gel where the EtBr/SYBR is no longer present (as it runs in the opposite direction to the DNA)
It's faint because it's less DNA. Gel staining dyes generally result in band intensities that are proportional to the mass of total DNA in the band, not the molar amount. Therefore, as Marcus says, if your insert is 10 times smaller than your vector, the liberated insert band will be ten times fainter even if they are present at the equimolar amounts that would be expected after 100% digestion.
EtBr does also migrate in the opposite direction of DNA, again as Marcus mentioned, but SYBR dyes don't - so this could be an issue depending on which stain you've used.
If you're feeling particularly industrious, and depending on how big your insert is, you might be able to resolve single-cut and double-cut fragments on-gel if you use a 30 cm 0.7% agarose gel and run it for 4-6 hours at 90V (make sure you also run single-cut control DNA). If you do this and see that all your plasmid fragments are indeed double cut, then you'll be confident that your digestions are going to completion.
Did you perform KpnI-mediated digestion first and then followed by SmaI-mediated digestion? or vice versa? An enzyme may inhibit the activity of an other enzyme. It is always good to deactivate the first enzyme prior to the second digestion.
Did you try fresh SmaI? Your enzyme might have lost its activity because of repeated freeze-thaw. You need to also check compatibility of the cut buffer for each enzyme.
Thanks to all.
The insert size= 8 kb, backbone= 4.5kb. I think here should be no issue like faint band. A linear vector is single fragment and is much much thicker than released fragments on double digestion.
Dear Alemu Tekewe@ i have performed first smaI and then kpnI.
Hmm...maybe your starting plasmid isn't as pure as you think. Maybe try re-transforming (or streaking out a new plasmid from glycerol stock if you have one) to colony purify the DNA.
Also with those sizes, you should be able to resolve single-cut and double-cut plasmid DNA on gel. If there's no ~12kb band after digestion, then your reaction went mostly to completion.
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