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Not getting good signal in sequencing


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#1 epigenetics

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Posted 14 July 2009 - 01:53 PM

Hello,
I need help in doing Bisulfite sequencing. For two genes, i did BS-PCR, Got single band, Purified using Qiagen Column, did cloning using invitrogen 2.1 vector, got good number of colony(did only ampicillin selection), grew overnight, then did miniprep and sent for sequencing.
I got back the result, The signal is too low. I really dont have any idea why i am not able to get my Vector seq atleast.
Please advise me what to do.

Thanks,

#2 lynnb

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Posted 17 July 2009 - 10:13 PM

Hello,
I need help in doing Bisulfite sequencing. For two genes, i did BS-PCR, Got single band, Purified using Qiagen Column, did cloning using invitrogen 2.1 vector, got good number of colony(did only ampicillin selection), grew overnight, then did miniprep and sent for sequencing.
I got back the result, The signal is too low. I really dont have any idea why i am not able to get my Vector seq atleast.
Please advise me what to do.

Thanks,


Questions for you:
Did you quantitate the the concentration and quality of the vector that you sent in for sequencing?
What was the concetration?
What was the A260/A280?
How much did you give them & how much did they use in their sequencing reaction?
What were the sequencing primers ?
It could be so many things.

Template quality (vector DNA prep) would be my first guess, but if the sequencing products were not properly precipitated after the sequencing reaction that would give poor data as well. If not enough DNA were put into the sequencing reaction that would give low signal too. Remember that if the sequencing group tell you they need a DNA template solution containing 250ng DNA they likely mean 250ng of your target DNA (what you want to sequence, the insert). If your vector is 2000bp and your insert were 1000bp you would give them an aliquot of 750ng of your vector construct which contains 250ng of your insert. If you had given them 250ng of your construct that would be 1/3 the amount of insert DNA & this could produce a low signal in sequence results. This is a common issue/mistake with people sequencing inserts contained in vectors.
lynn

lynn

#3 epigenetics

epigenetics

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Posted 20 July 2009 - 12:12 PM

Hello,
I need help in doing Bisulfite sequencing. For two genes, i did BS-PCR, Got single band, Purified using Qiagen Column, did cloning using invitrogen 2.1 vector, got good number of colony(did only ampicillin selection), grew overnight, then did miniprep and sent for sequencing.
I got back the result, The signal is too low. I really dont have any idea why i am not able to get my Vector seq atleast.
Please advise me what to do.

Thanks,


Questions for you:
Did you quantitate the the concentration and quality of the vector that you sent in for sequencing?
What was the concetration?
What was the A260/A280?
How much did you give them & how much did they use in their sequencing reaction?
What were the sequencing primers ?
It could be so many things.

Template quality (vector DNA prep) would be my first guess, but if the sequencing products were not properly precipitated after the sequencing reaction that would give poor data as well. If not enough DNA were put into the sequencing reaction that would give low signal too. Remember that if the sequencing group tell you they need a DNA template solution containing 250ng DNA they likely mean 250ng of your target DNA (what you want to sequence, the insert). If your vector is 2000bp and your insert were 1000bp you would give them an aliquot of 750ng of your vector construct which contains 250ng of your insert. If you had given them 250ng of your construct that would be 1/3 the amount of insert DNA & this could produce a low signal in sequence results. This is a common issue/mistake with people sequencing inserts contained in vectors.
lynn

lynn



Thanks for the reply, but it seems it is quite different whatever my core facility said to me.
My vector is 3.9 KB and insert is 200 bp, so total 4.1 KB, so they said to send 410 ng DNA with 3.2 pm of any primer. I used M13 Forward Universal primer.and my vector concentration is 25 ng/microlt. I generally use 1:3 ratio during cloning.
well, with 410 ng DNA when i got low signal, my facility said to double DNA amount keeping Primer same, But result is much worser than before.
No clue whats happening.




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