T4 kinase in end repair protocol

klytie · 07-06-2011, 06:22 AM

Hi!
I'm new in this, so forgive me if my question is stupid. Why do I need to add T4 PNK to the mixture of Klenow and T4 polymerase when repairing the ends of enzyme fragmented library. I'm trying to put together a protocol for Illumina sequencing but at the moment we are not using NEB next or Illumina or any kind of kit prepared for end repairing. There are only separately bought enzymes on the menu. If I remember correctly, I have seen protocols with and without T4 PNK. When do I need to use this enzyme and when is it not necessary to put thisone in? Can anyone also provide me the units per end repair reaction for T4 pol and Klenow?

Many thanks!

pmiguel · 07-06-2011, 09:04 AM

If I remember correctly, T4 PNK will add a phosphate to a hydroxyl 5' end and/or remove a phosphate from the 3' end of a DNA or RNA strand. This is the desired phosphate configuration for most ligases.

What is the end produced by the enzyme being used to fragment the library? Typically a restriction enzyme will give 5'-phosphate/3'-hydroxyl that is needed. But the overhangs may or may not be compatible.

You can get the number of units necessary per mole of your fragment ends from the web site of the manufacturer you order the enzyme from.

--
Phillip

klytie · 07-06-2011, 11:00 PM

Thank you for your answer. The enzyme is DNAseI and following end repair comes as usual A-tailing with Klenow exo-. So...I'd better use those three enzymes just to be sure my reactions work?

seq_me · 07-06-2011, 11:45 PM

Yes t4 polymerase will make the ends blunt and T4 PNK will make sure that 5' ends are phosphorylated and 3' ends have the hydroxyl group. I dont think of a reson of not using this esp if your DNA fragments are generated using sonication

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07-06-2011, 06:22 AM	#1
klytie Junior Member Location: Tallinn Join Date: Jul 2011 Posts: 5	T4 kinase in end repair protocol Hi! I'm new in this, so forgive me if my question is stupid. Why do I need to add T4 PNK to the mixture of Klenow and T4 polymerase when repairing the ends of enzyme fragmented library. I'm trying to put together a protocol for Illumina sequencing but at the moment we are not using NEB next or Illumina or any kind of kit prepared for end repairing. There are only separately bought enzymes on the menu. If I remember correctly, I have seen protocols with and without T4 PNK. When do I need to use this enzyme and when is it not necessary to put thisone in? Can anyone also provide me the units per end repair reaction for T4 pol and Klenow? Many thanks!

07-06-2011, 09:04 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	If I remember correctly, T4 PNK will add a phosphate to a hydroxyl 5' end and/or remove a phosphate from the 3' end of a DNA or RNA strand. This is the desired phosphate configuration for most ligases. What is the end produced by the enzyme being used to fragment the library? Typically a restriction enzyme will give 5'-phosphate/3'-hydroxyl that is needed. But the overhangs may or may not be compatible. You can get the number of units necessary per mole of your fragment ends from the web site of the manufacturer you order the enzyme from. -- Phillip

07-06-2011, 11:00 PM	#3
klytie Junior Member Location: Tallinn Join Date: Jul 2011 Posts: 5	Thank you for your answer. The enzyme is DNAseI and following end repair comes as usual A-tailing with Klenow exo-. So...I'd better use those three enzymes just to be sure my reactions work?

07-06-2011, 11:45 PM	#4
seq_me Junior Member Location: San Francisco Join Date: Feb 2010 Posts: 8	Yes t4 polymerase will make the ends blunt and T4 PNK will make sure that 5' ends are phosphorylated and 3' ends have the hydroxyl group. I dont think of a reson of not using this esp if your DNA fragments are generated using sonication