KpnI and BamHI double digest help - (Apr/24/2006 )
I'm trying to do cloning but the thing is somehow, i'm unable to cut my insert with this 2 RE. NEB recommend its buffer 2 but somehow it doesnt work for me...
and fermentas said that they are not compatible and dun share the same buffer. KpnI require less salt while BamHI needs high salt conditions.
any ideas to help me?
and fermentas said that they are not compatible and dun share the same buffer. KpnI require less salt while BamHI needs high salt conditions.
any ideas to help me?
well, actually NEB recomends to do sequential digest, if using KpnI and BamHI.. if you're having problems with double digestion, i would just try doing one after the other..
Use Fermentas BamHI buffer and add double amount of KpnI compared to BamHI, so if you are using 0.5 microliter of BamHI for reaction, use 1microliter of KpnI in the digestion and incubate at 37C.
http://www.fermentas.com/doubledigest/
Check if you have enough number of overhangs near restriction site if you are working with linearised DNA. You need at least 4 nucleotides besides restriction site so that restriction enzyme sits on top of it to the job. If not, clone in it polyA vactor like Topo TA and cut it through that plasmid.
well, actually NEB recomends to do sequential digest, if using KpnI and BamHI.. if you're having problems with double digestion, i would just try doing one after the other..
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actually i have tried this method already but somehow it didnt work for me. I cut with KpnI first for 1.5hrs and then top up the buffer with KCl and mercaptoethanol to a final conc that simulate the BamHI buffer. I add the BamHI and incubate for another 1.5hrs but it didnt work as good as i expected..maybe about 50% or less cut only.
Hi,
Normally when I need to do a double digestion, I realised my digestion in two step.
1) Cut your DNA with the enzyme n°1 in a tube and with the enzyme n°2 in a second tube. Like that you can be sure that each enzyme cut well.
2) When it's done and ok, realise the second step of digestion on your first digestion product but with the second enzyme.
It's always works for me!
biofred
http://www.fermentas.com/doubledigest/
Check if you have enough number of overhangs near restriction site if you are working with linearised DNA. You need at least 4 nucleotides besides restriction site so that restriction enzyme sits on top of it to the job. If not, clone in it polyA vactor like Topo TA and cut it through that plasmid.
I do have 4 nucleotides besides the restriction sites so it should be able to sit and cut well.
I'm using TA cloning at the moment and hopefully it works as there are a few factors i'm worrying about:
1)PCR size is 3.2kb
2)PCR band is not very intense and I dun how much I hve collected after I gel extracted and purifed.
3)Unable to further amplify when i run a 2nd PCR.( i get smear from the top.)
3)Unable to further amplify when i run a 2nd PCR.( i get smear from the top.)
May be stupid question but are you using enough of Taq? I had same issue. My background is not molecular biology so I had read everything in theory and when I started working at lab bench, I was told to use half amount each of Taq and Pfu but this was in terms of amount and not units. I used to get very little PCR product as I used to use very little Taq and more of Pfu. Taq is faster in doing job than Pfu. So if you increase amount of Taq, you may get better PCR product, at least I did.
I have noticed same, you lose a lot in Gel extraction of PCR purification so it is very essential if you are planning to do two step digestion. Try one digestion with two enzymes as Fermentas recommends on their website. It helps with me for some other enzyme combination.