DNA blunting - DNA blunting troubleshoot (Aug/16/2008 )

Hi Dear friends....
I need your help.
Everybody in my lab has been falling the last 3 weeks
to succeed a blunting reaction. We troubleshoot but
none of them seem to be the problem. We also buy a
new kit but the problem remains.
And now, I need to do a blunting reaction next week,
can someone give me an idea of all possible troubleshoots
during DNA blunting, plz?
If you have had the same experience too, tell me how you
had solved it.
Thanks for always being there, guys.

If you mention your blunting condition and strategy, it will be easier to troubleshoot it.

You do need to list exactly what you have done. There are quite a few things that could have gone wrong.

The amount of DNA, size and molar ratio you are using is also useful.

Has anybody been having trouble ligating sticky end inserts in the lab? It is possible that your T4 ligase or T4 ligase buffer has gone bad. Have you tried to run out some of your ligation product onto a gel to see if the ligation reaction has been successful?

Where does the blunt end insert come from? PCR or digest?

Why are you using a blunt end ligation strategy? Is it possible to use a sticky end ligation?

Is the quality of your insert and vector okay? Have ran a gel to check that they are okay?

If you must use blunt end ligation, using quick ligase (addition of PEG6000 10% final) is helpful.

How long are you ligating for? Increasing the duration and lowering the temperature (16 Celsius or 4 Celsius) can sometimes help.

Did you dephosphorylate your vector? Since this is a blunt end ligation you need to dephosphorylate the vector. Alternatively can you skill your vector? Ie if the vector was blunt ended using EcoRV, recirculisation of the vector without the insert will regenerate the EcoRV site, so kill the ligation mix with EcoRV can reduce the back ground.

Did you do any controls once you transformed your ligation mix? Vector only, insert only, vector+insert. How many colonies did you recover?

Hallo I used Klenow for blunting ends but not succes came. It was because Klenow has polymerisation activity and If you let him enoug time and has not what to do it will conects ends of oposite strands
I have good experiences with, S1 nuclease and recently Fermentas has product called Blunting enzyme and me frend has good results with this, so try that
good luck

klenow blunt end 3' overhangs and if provide dNTP fills in 5' overhang.
S1 nucleases blunt ends 5'overhangs.