Klenow - ligation problem - Can Klenow add some extra nucleotides.. (Jan/03/2008 )

Hello to everyone!
I have trouble with blunt end ligation, and I've read loads of pages today on this forum so I see I'm not the only one with that problem.. but still I have some extra questions:

Can Klenow add some extra nucleotides when filling recessed 3'-termini, if a reaction is done at 37°C for 1 hour, and the protocol says 10 minutes ?!

I need to ligate insert cut with BamHI (5' overhang) and ScaI (blunt end) so I use Klenow (Fermentas) to blunt this 5' end, but I don't get very good ligation result. I have checked the ligase, it works fine on lambda/HindIII..

Since i can't check Klenow by running insert on gel, is there another way to do that, and is the reaction time relevant for Klenow to do job – can extra time of incubation do some 'damage' or it doesn't matter?

Thanks!

I would be careful with this enzyme and would definitely not overincubate it with my DNA. I think it still contains 3'-5' exonuclease activity so it can eat your DNA rather than add extra bps. I would try it again with a short incubation. Look in the New england Biolabs book, as they have good info that worked for me with klenow in the past.

we have used klenow from neb and they have protocol for it. Follow it and it will work.

thank u both for quick reply!

@Mountainman
Yes, you're right, it could eat it and that would also be a problem for my ligation..
I've read NEB instructions and they are a bit different from Fermentas
NEB: Incubate 15 minutes at 25°C
Fermentas : Incubate the mixture at 37°C for 10 minutes

Anyway, it seems smart not to overincubate, as you said.

@scolix
I would, but we only have Fermentas enzyme and buffer in lab, so we play at their rules (except the time)..

I'll try it differently, so we'll see!

C. Joyce (1984 BRL Focus 6(1): 6) cautions that the use of more enzyme, longer reaction times than 10-15 minutes, or temperatures higher than room temperature which promote breathing may decrease the yield of filled-in product. Klenow’s 3’ > 5’ exonuclease activity removes the final nucleotide after the fill-in reaction. Alternating polymerase and 3’ > 5’ exonuclease activity eventually convert all of the terminal dNTP to dNMP which results in the loss of the terminal nucleotide. The exo- version of the enzyme is not recommended for fill-in reactions prior to DNA ligation, as extra nucleotides added to the 3’ end of a blunt-end DNA will not be deleted by the exonuclease activity of the intact enzyme.

C. Joyce (1984 BRL Focus 6(1): 6) and del Solar and Espinosa (1991 NAR 19 (8) 1956) suggest that some restriction enzymes stay attached to the cut ends of molecules and may require phenol:chloroform extraction (the most frequent offenders are BamH I, Hind III and Pst I) prior to Klenow fragment fill-in.

DO NOT heat inactivate, as this accelerates the 3' > 5' exonuclease activity.

OR: Stupidly simple
Order primers, amplify the fragment from the plasmid with a polymerase that doesn't add any NTs (such as Pfx, Pfu,..) and blunt ligate it into your vector. Works for me every time.
I know it's not the same thing, but sometimes it's rewarding to try a different approach - especially if you have been struggling with this Kenow one for quite some time now..

This is the third time I do this, and it seems that's it's going to be made in the same way as it was when I did it before (which so far, didn't have brilliant result) unless I think of another solution or perhaps find a point where it fails. This time, I'm using all of my isolated plasmid-DNA..

Here is what I do, in brief:

After restriction I use NH4Ac/EtOH for DNA precipitation to get rid of enzymes and their buffers, from which i get a mixture of few plasmid fragments that contains the fragment I need. Then I do another restriction (which is just to digest and remove a fragment similar in size with 'the one I need' and make geneclean easier.. )
After that restriction I just add Klenow (it works in that buffer + I add some of Klenow-buffer) and dNTPs in restriction mixture. (I need Klenow just for my fragment so the rest doesn't bother me..) Incubation on 37°C, 1 hour. I inactivate Klenow by adding EDTA and incubating it for 30 min on 70°C.
Then: ethanol precipitation, gel extraction and that is the moment at which I hope I will have desirable fragment, blunt and ready for blunt-end ligation! But after ligation, I run the gel and it doesn't look good. First, it seems I've lost a lot of my DNA (because of many extraction protocols) and also very little gets ligated

So after a lots of consideration, I thought the step with Klenow could be the one to blame..

Btw, I work in lab on a tight budget, so I think that options which include ordering new stuff may be hard to expect.. and I'm not in charge..

Thanks for all the help and suggestions
I hope you continue with them because they have all been such a great help to me!