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sequencing results


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#1 biologist82

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Posted 07 October 2014 - 04:34 AM

Hi,

 

I have 2 sequencing results at attachment. They are different PCR products and was used same primers. For 2 samples after PCR purification with exosapit, below protocol was used:

 

Big Dye v3.1: 2 ul

Buffer: 1 ul

primer: 1 ul from 3.2 pmol

pcr: 1 ul

water: 5 ul

 

Sephadex was used.

 

Sequence machine is 3130. Pop7 and buffers are fresh.

 

Samples were checked with agarose gel electr. after PCR, and their bands are good and one.

 

But my sequence results are not very good.

 

What is your recommendations about this results?

 

Thanks for your help

 

Regards

 

 

Attached Thumbnails

  • seq1.JPG
  • seq2.JPG


#2 phage434

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Posted 07 October 2014 - 05:21 AM

I can't tell much without looking at an electropherogram. The most likely cause is either too much or too little template. I'd vote for too much.



#3 phage434

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Posted 07 October 2014 - 05:28 AM

I should also mention that almost all sequencing is done with dramatically less BigDye. You can probably get perfectly good results reducing the BigDye amount to 0.5 ul and increasing the water to compensate. This might save you some money.



#4 biologist82

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Posted 07 October 2014 - 07:58 AM

What is the ideal template (pcr product) amount for total 10 ul volume?



#5 phage434

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Posted 07 October 2014 - 12:09 PM

I would use 10 ng or less in the reaction. Too much is more damaging than too little, typically. Your sequencer manual probably has good advice.  It has been a long time since I set up this reaction, so I would read more before taking my advice.

http://openwetware.o..._for_Sequencing






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