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long range PCR - purification of pcr frogament 9-13kb (Sep/22/2009 )

Hello,

I have to amplify a 30kb fragment . I decided to do 3 overlapping amplifications. I bought Roche long PCR kit. I was not sure if I needed nested PRIMERS, I wrote to the technical team and they said they used a single round. My PCR is negative, do you think I should design nested primers? what would you suggest?

2nd problem, has anyone used a purification kit for fragments more than 10kb? (9.5-13kb)?

Cheers,
belle

-trim5-

Before you go and design new primers, why don't you give us your PCR protocol (ie cycle temps and times, reaction volumes, etc)- there are many things that could be going wrong other than your primers, and it would be a shame to spend money on new ones when just tweaking your protocol could do the trick.

-leelee-

leelee on Sep 23 2009, 02:17 AM said:

Before you go and design new primers, why don't you give us your PCR protocol (ie cycle temps and times, reaction volumes, etc)- there are many things that could be going wrong other than your primers, and it would be a shame to spend money on new ones when just tweaking your protocol could do the trick.

Hi Leelee thanks for replying, I have the protocol below, which is the one they recommend, comes with the kit...
master mix 1
Reagent vol
Nuclease free water 17
dNTP mix (10mM each) 2.5
T5A GFP1 (10M) 1.5
T5A GFR1 (10M) 1.5
Mix volume 22.5
Aliquot 22ul of the mix in each tube, then add
DNA (~50ng/ul) 2.5
Final vol 25
Mix by pipetting up and down, DO NOT VORTEX. Centrifuge briefly

Make mix 2 by adding the following reagents below in a thin-walled tube on ice:
Reagent Volume (L)
Nuclease free water 19.25
10X buffer (27.5mM MgCl2) 5 two buffers one with detergents one without : used both, both negative, one with detergent has smears
1→Enzyme mix (5U/ul) 0.75
Mix volume 25
Mix by pipetting up and down, centrifuge briefly

Add mix 2 to 1(final vol=50μL), Mix by pipetting up and down and place immediately in thermocycler.
Cycling conditions
92C 2 min 1X
92/57/68 C 10s/30s/10m 10X
92/57/68 C 10s/30s/10m +20sec/cycle 25X
68C for 7min HOLD 4C

Look forward to hearing from you.

All the best,
B

-trim5-

So nothing really jumps out at me with your protocol. I don't have a lot of experience with amplifying large products though- in fact the largest I have had to amplify is just 4kb. However, that did require a bit of tweaking to get right, with my extension time, which in your case seems to be appropriate.
Have you tried different annealing temperatures?? Maybe you could do a gradient or touchdown PCR??
Another thing- have you tried varying dilutions of your template??

-leelee-

leelee on Sep 24 2009, 03:03 AM said:

So nothing really jumps out at me with your protocol. I don't have a lot of experience with amplifying large products though- in fact the largest I have had to amplify is just 4kb. However, that did require a bit of tweaking to get right, with my extension time, which in your case seems to be appropriate.
Have you tried different annealing temperatures?? Maybe you could do a gradient or touchdown PCR??
Another thing- have you tried varying dilutions of your template??

Thanks lee lee,

I am trying a touch up PCR now, will let you know how it turns out, if all fails, I will design, a primer to use in a hemi-nested PCR.

Thank again!!!

Belle

-trim5-

If you're amplifying 30 kb, I would think even 10 minutes extension might not be enough. Data from Roche of the rate for their enzyme mix suggests 20 minutes for 30 kb.

As for purification kits, we have used the Promega Wizard SV for 9 - 10 kb, no worries.

-swanny-

I agree with Swanny, extension time is rather short for 30kb.

also, have you looked at varying the annealing temperature. You might want to test a few annealing temperatures.

do you know if your primers uniquely bind to your template region.

also, have you looked at your template sequence? Anything undesirable such like long stretches (>1kb) of dinucleotide repeats? These kind of DNA sequences are hard to amplify.

anyone used a purification kit for fragments more than 10kb? (9.5-13kb)?

Yup. Does it work? kind off, you lose alot of DNA. But if you have a lot to begin with you can usually get by. I believe there are DNA purification kits that are made to handle fragments larger than 10kb. I have not used those before.

Alternatively you can use gel electroelution and that works. Depending on your set up you might need to butanol dehydrate your DNA sample and that can be time consuming.

-perneseblue-

swanny on Sep 28 2009, 05:53 AM said:

If you're amplifying 30 kb, I would think even 10 minutes extension might not be enough. Data from Roche of the rate for their enzyme mix suggests 20 minutes for 30 kb.

As for purification kits, we have used the Promega Wizard SV for 9 - 10 kb, no worries.


Thanks Swanny, I am amplifying it in 3 overlapping fragments of 12 kb, so each reaction is for about 12kb.

Thank for the reply.

-trim5-

I'd still go with a single reaction, because you reduce your workload of having to stitch the three products together.

Also, when the DNA is that big, I'd consider just doing an EtOH precipitation. Your primers and other junk won't come down, so you end up with clean DNA. If you need to get rid of the template, add 1 ul of DpnI after the PCR and incubate for 15 minutes, then precipitate (obviously the template is only fragmented, but it should stop it interfering downstream...).

-swanny-

swanny on Sep 29 2009, 07:09 AM said:

I'd still go with a single reaction, because you reduce your workload of having to stitch the three products together.

Also, when the DNA is that big, I'd consider just doing an EtOH precipitation. Your primers and other junk won't come down, so you end up with clean DNA. If you need to get rid of the template, add 1 ul of DpnI after the PCR and incubate for 15 minutes, then precipitate (obviously the template is only fragmented, but it should stop it interfering downstream...).


This sounds good, thanks Swanny

-trim5-