pWM91 cloning problem - (Jul/28/2011 )

I have been trying to clone my 4.1kb PCR product (flanked with BamHI site) into pWM91.

I treated them with BamHI and did ligation at 1:3 vector:insert ratio. I kept the total DNA concentration below 150ng in a reaction.

So far I did not get anything.

The primer design is something like this : 5' CGGGATCCCGNNNNNNNNNNNN 3'.

Will my PCR product cause any problem if I cut it with BamHI?

I usually put CGCGGATCC, and never had any prob in complete digestion. Another thing i noticed in your primer is the overhang CG is also at at the 3' end, i dnt know is this also the case in your primer design or Typo error or you representing both primers overhang..though this has nothing to do with digestion but would alter the frame of your insert.
if you post the complete protocol of your cloning may be ppl here would help you to troubleshoot...

badguy on Fri Jul 29 02:19:47 2011 said:

Thanks for the reply. The CG at 3' is not a typo. I'm just following the guide from NEB. I'm a newbie in primer design

The BamHi restriction protocol is as below:

500ng of DNA

1X BamHI buffer

5U BamHI

ddH2O: top up to 20ul

Incubation at 37C for 1 hr, heat inactivate at 80C for 20min.




Ligation protocol is as below:

Vector - 50ng

Insert - 75ng

1X NEB Ligase buffer

0.5ul of T4 DNA Ligase

ddH2O - top up to 10ul.

Incubated at room temp (~23C) for 30min

So you have added the overhang CG at both the ends of the RE site, Be aware that since you have added two bases at the 3' end, it would alter the reading frame of your insert if your downstream application of the construct is for expression, neednt bother much if you are going to use the construct for other applications.
Coming to your protocol.., how long you digested the vector (i would suggest a minimum of 3 hrs at 37), think you are using the NEB Enzymes for digestion and ligation, so you neednt have to heat inactivate postdigestion, just purify the digested vector after digestion (Gel purification is recommended), you got to dephosphorylate the vector after this (max 1 hr at 37 with CIP ), after dephosphorylation i again use to purify the vector (here i do PCR purification)..thats about Vector ..
Then insert, same digestion like Plasmid and without heat inactivating you can purify using any PCR purification Kit...now check 1 micolitre of both vector and insert on agarose (i quantify based on the gel) but you can quantify using nanodrop and proceed..
Then regarding Ligation i would suggest you to do overnight at 15 degrees...

Anothr thing is the control, you got to use atleast a vector alone ligation to see how much background your prepared vector gives on transformation.

Finally even if all these are good, you would fail if your competent cells are not enough efficient...so have a check over this as well...

good luck...

badguy on Fri Jul 29 09:10:16 2011 said:

Thanks for the reply. The CG at 3' is not a typo. I'm just following the guide from NEB. I'm a newbie in primer design

The BamHi restriction protocol is as below:

500ng of DNA

1X BamHI buffer

5U BamHI

ddH2O: top up to 20ul

Incubation at 37C for 1 hr, heat inactivate at 80C for 20min.




Ligation protocol is as below:

Vector - 50ng

Insert - 75ng

1X NEB Ligase buffer

0.5ul of T4 DNA Ligase

ddH2O - top up to 10ul.

Incubated at room temp (~23C) for 30min

GNANA on Fri Jul 29 10:58:25 2011 said: