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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 300┬Ál 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 sad.gif

-ivanadj-

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.

-swanny-

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 sad.gif

Thanks

-ivanadj-

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. huh.gif

Thanks!

-ivanadj-

QUOTE (ivanadj @ Jun 18 2008, 05:48 PM)
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. huh.gif

Thanks!

I wasthinking of the insert, but you can do the same expt for the vector as well, just don't try to run them on the same percentage gel! biggrin.gif

-swanny-