For Nextera mate-pair kit, Is it possible to reduce the tagmentation reaction time?

ychang · 03-05-2013, 04:39 PM

For Nextera mate-pair kit, Is it possible to reduce the tagmentation reaction time to get more long fragment, say 10kb? we have use this kit and found most of the smear is around2-7kb, but we want to do a large insert lib. does anyone try to reduce the tagmentation reaction time to get more long fragment, say 10kb? Thanks.

ZWB · 03-05-2013, 04:46 PM

Have you tried using less enzyme? I think using less transposon would be a more effective way to increase fragment size than decreasing (the already short) reaction time.

03-06-2013, 09:54 AM

I'd say this LARGELY depends on the quality of your input DNA. Usually with very intact HMW DNA we've got a smear from 3-12kb and above. One of the samples, with slightly fragmented smeary DNA, was completely digested to below 1kb under the very same conditions as the samples mentioned previously.

MesutOezil · 03-24-2014, 06:03 AM

We tried reduced amounts of tagmentation enzyme (4 ul or 8ul instead of 12 ul), expecting longer fragments, but it did not make any difference, unfortunately.

This does not help the yield of relatively long tagmented DNA, but may help many users in terms of budget. But, probably we also need to know how to prepare self-made tagmentation buffer.

Katerina Cvikova · 04-16-2014, 12:08 AM

Hi, I would like to ask if you sorted out your problem. I'm planing to use a Nextera Mate Pair Gel-Plus Sample Preparation kit for mate pair library. The problem is, that I don't have intact HMW DNA but only DNA from sorted and amplified wheat chromosome where the size is about 20 kbp. So I am interested if the decrease of time or amount of enzyme assure the less fragmentation but good tagging by adapters. Thanks. Katka

MesutOezil · 04-20-2014, 07:16 AM

Hi Katka,
We tried only reduced tagmentation enzyme amounts (4 and 8 ul instead of 12ul), and it made some difference. We tested two species, and it seemed that different species show different effects upon varying the tagmentation condition. It is also of note that size distribution of tagmented DNA fragments should be measured in a reliable method.

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03-05-2013, 04:39 PM	#1
ychang Member Location: philly Join Date: Oct 2010 Posts: 17	For Nextera mate-pair kit, Is it possible to reduce the tagmentation reaction time? For Nextera mate-pair kit, Is it possible to reduce the tagmentation reaction time to get more long fragment, say 10kb? we have use this kit and found most of the smear is around2-7kb, but we want to do a large insert lib. does anyone try to reduce the tagmentation reaction time to get more long fragment, say 10kb? Thanks.

03-24-2014, 06:03 AM	#4
MesutOezil Member Location: Japan Join Date: Feb 2011 Posts: 12	Reducing tagmentation enzyme We tried reduced amounts of tagmentation enzyme (4 ul or 8ul instead of 12 ul), expecting longer fragments, but it did not make any difference, unfortunately. This does not help the yield of relatively long tagmented DNA, but may help many users in terms of budget. But, probably we also need to know how to prepare self-made tagmentation buffer. Last edited by MesutOezil; 03-30-2014 at 12:42 AM.

03-05-2013, 04:46 PM	#2
ZWB Member Location: Livermore, CA Join Date: Apr 2011 Posts: 27	Have you tried using less enzyme? I think using less transposon would be a more effective way to increase fragment size than decreasing (the already short) reaction time.

03-06-2013, 09:54 AM	#3
lorendarith Guest Posts: n/a	I'd say this LARGELY depends on the quality of your input DNA. Usually with very intact HMW DNA we've got a smear from 3-12kb and above. One of the samples, with slightly fragmented smeary DNA, was completely digested to below 1kb under the very same conditions as the samples mentioned previously.

04-16-2014, 12:08 AM	#5
Katerina Cvikova Junior Member Location: Olomouc Join Date: Apr 2014 Posts: 6	Hi, I would like to ask if you sorted out your problem. I'm planing to use a Nextera Mate Pair Gel-Plus Sample Preparation kit for mate pair library. The problem is, that I don't have intact HMW DNA but only DNA from sorted and amplified wheat chromosome where the size is about 20 kbp. So I am interested if the decrease of time or amount of enzyme assure the less fragmentation but good tagging by adapters. Thanks. Katka

04-20-2014, 07:16 AM	#6
MesutOezil Member Location: Japan Join Date: Feb 2011 Posts: 12	Hi Katka, We tried only reduced tagmentation enzyme amounts (4 and 8 ul instead of 12ul), and it made some difference. We tested two species, and it seemed that different species show different effects upon varying the tagmentation condition. It is also of note that size distribution of tagmented DNA fragments should be measured in a reliable method.