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Old 03-27-2012, 06:49 AM   #1
sanju0891
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Unhappy Short fragments removal- Amplicon sequencing

Hi
i have performed amplicon sequencing using gs junior and in the first run they were peaks around 120-150 bp.

so in the next run i want to reduce these peaks. i did 2x purification with the ampure beads(0.8: 1 ratio) but still there are slight or faint bands visible in agarose gel. How do i get rid of these bands completely?

if i am purifying it more further i am losing my ampliocns which are around 300bp.

Please help me.

Thanks
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Old 03-28-2012, 07:49 AM   #2
pmiguel
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You might be able to drop to a lower ratio. Depends on your lot of beads. If you take a DNA ladder of some sort, you can calibrate your batch of beads by trying different ratios. Here is an example:



For this calibration, it looks like the best differentiation between 300 and 150 bp would be around 0.7:1 or 0.75:1.

Alternatively you can just cut your band of a desired size out of a gel and extract the DNA from the slice. But that may be more work. Well, maybe not for a single band. But if you had numerous samples it would be.

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Old 03-28-2012, 08:41 AM   #3
sanju0891
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Hi
Thankyou so much for replying

with the 0.8:1, i was able to remove all bands below 250bp , eventhough there is a slight loss of my amplicon samples.

but now the problem was with the quantitation of the samples. we used the Nanodrop 2000 to measure the conc of DNA in the purified samples. but the nanodrop values are negative for concentration of DNA.

i do not understand this and i am confused with wat procedure shuould i use to quatify my samples?
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Old 03-28-2012, 09:36 AM   #4
pmiguel
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A nanodrop spectrophotometer is not useful for quantifying samples below 20 ng/ul or so. Use a fluorimeter, a bioanalyzer chip, and/or your gel. Or qPCR.

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Old 03-29-2012, 03:40 AM   #5
sanju0891
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Thank you for the reply.

we are thinking to use the picogreen assay fro the quantitation.
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Old 03-29-2012, 05:31 AM   #6
HMorrison
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We use picogreen routinely, then make amplicon pools and quantitate the pools with Kapa qPCR kit. Has been very reliable.
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Old 03-29-2012, 05:35 AM   #7
sanju0891
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i was just reading all the threads and evrybody recomends Kappa kit.

i have a small question regarding this. which kit from the KAPA biosystems is best fro GS junior titanium- LIB A
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Old 03-29-2012, 06:43 AM   #8
HMorrison
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Default Kapa kits

I don't know all the variations and it depends on your cycler; check with the company first if you have trouble sorting out their web site (really needs a decision tree).

We use the FLX version, not the Ti version, with LibA (adapters for LibA/Titanium are like old FLX adapters, not like LibL/Titanium shotgun). Part number is KK4830. This is for ABI Prism qPCR instrument. We have a standard GS-FLX, not Junior.
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