pcr,ligation and transformation with RE SalI and XhoI - (Jun/17/2008 )

Hi to evrebody,

I hope somebody will have advice for my problem:

I did PCR with 2 RE :SalI and XhoI.I checked pcr product with other RE and it is ok.Than with same RE I cutted the vector (IBA3+,IBA7).Digestion was overnight,and I also tried with 1 hour digestion.
After digestion I did enzyme inactivation, gel extraction,ligation with T4 ligase(tried 22C o/n,16C o/n,22C 1h),enzyme inactivation,than drop dialisys and electrotransformation,after transformation I incubated ib 300µl LB for 1h and plate on plate with antibiotic.
Controls are:
1)without insert
2)without ligase and vector

Result:
Reactin plate is full
C1 plate is empty
C2 plate once was empty and once full as reaction plate.

Also I tried to treat the vector with SAP one hour,but as you see no good result.

So has anybody any idea how to solve the problem?

Thanks

Check out the fine print on Sal I in the NEB website. It doesn't like PCR products. Why? Don't know. Best suggestion is to do a PCR cleanup.
Also, XhoI doesn't like supercoiled DNA, so that might be affecting your plasmid digestion.

To check your digestions are working properly, take some of the digests and heat-kill the RE. Then do a short ligation reaction and run a gel. If you don't have a ladder or a significntly higher band, one of your enzymes isn't working properly. A double-size band means only one enzyme is working, a ladder that is skewed towards double (rather than triple and quadruple-size) means one of the enzymes isn't working well, so digest with an excess of that enzyme.

Thanks for sugestion

I know that these two enzymes are very problematic,but this is the only posibility for cloning,because the sequence doesnt allow other combintion

Thanks

QUOTE (swanny @ Jun 18 2008, 01:17 AM)

Check out the fine print on Sal I in the NEB website. It doesn't like PCR products. Why? Don't know. Best suggestion is to do a PCR cleanup.
Also, XhoI doesn't like supercoiled DNA, so that might be affecting your plasmid digestion.

To check your digestions are working properly, take some of the digests and heat-kill the RE. Then do a short ligation reaction and run a gel. If you don't have a ladder or a significntly higher band, one of your enzymes isn't working properly. A double-size band means only one enzyme is working, a ladder that is skewed towards double (rather than triple and quadruple-size) means one of the enzymes isn't working well, so digest with an excess of that enzyme.

Do you mean to check insert (pcr product) or vector.

Thanks!