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Old 06-15-2016, 05:03 AM   #1
emarsola10
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Location: Brazil

Join Date: May 2016
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Question Pippin-Prep - External or Internal markers?

Hi Seqanswers community,

I going to test size selection step in Pippin-Prep and Im not sure about the advantages and disadvantages to use the external or internal markers.

I going to use a 2% dye-free Pippin-Prep cassete gel to select two different ranges, 200-250 and 400-450 bp. The marker options for my experiment are Marker L (internal standards) or Marker K (external standards).

I have already performed a test using the external Marker K to select the above mentioned ranges and using the 'Range Program Mode'. However, the product size recovered was smaller than selected (around 150 bp for the 200-250 range and around 350 for the range 400-450). I guess if I use a internal instead external marker I can improve accuracy, but I afraid that I can collect a marker fragment togheter with the recovered product.

Thank you.
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Old 06-15-2016, 05:49 AM   #2
kmcarr
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Quote:
Originally Posted by emarsola10 View Post
Hi Seqanswers community,

I going to test size selection step in Pippin-Prep and Im not sure about the advantages and disadvantages to use the external or internal markers.

I going to use a 2% dye-free Pippin-Prep cassete gel to select two different ranges, 200-250 and 400-450 bp. The marker options for my experiment are Marker L (internal standards) or Marker K (external standards).

I have already performed a test using the external Marker K to select the above mentioned ranges and using the 'Range Program Mode'. However, the product size recovered was smaller than selected (around 150 bp for the 200-250 range and around 350 for the range 400-450). I guess if I use a internal instead external marker I can improve accuracy, but I afraid that I can collect a marker fragment togheter with the recovered product.

Thank you.
I have used the Blue Pippin to isolate fragments in the 175-250bp range using Dye-Free cassette BDF2010 (2%) with internal standard V1 and it has worked very reliably. The internal standards are very small so they do not co-migrate with your 200bp fragments.

You said you tested using Marker K as an external standard. Looking at the Pippin Cassette Reference Chart it lists Marker K as an internal standard meant for the 1.5% cassette. The external standard designated for 2% cassettes is Marker E. That may explain the discrepancy in the fragment sizes recovered.
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Old 06-15-2016, 05:53 AM   #3
luc
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The internal standards have shorter fragment lengths; they will not be eluted with your sample. With internal standards you can run 5 samples per cassette. We have not noticed any principal differences in accuracy.
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Old 06-15-2016, 04:12 PM   #4
emarsola10
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Default Pippin-Prep - External or Internal markers?

Thank you for reply.
I used the external marker E (CEF 2010) and not K, as I said before.

I think that people from SAGE assistance has found the problem. Checking the log files of our run they found a hump in the curves of the current profile for all lanes, whereas the expected curve must be flat. They gave the following tip: "This hump typically indicates that the input sample contains excess salt (>80mM ionic strength). High salt causes an altered migration, and can cause inaccurate sizing. I would suggest performing a purification step to remove excess salt before loading future samples on the Pippin Prep.". This makes sense because we entered with a concentrate PCR product in the gel.
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