3 replies to this topic

[contactsports]

My experience with sequencing basically involves everything necessary to get DNA prepped for sequencing and then sending it away to get sequenced.  I also have only worked exclusively with Drosophila.  Now, I work for a start-up with limited funds, so I am trying to get this working without having to buy too many more reagents/machines.

Background: We are trying to sequence SNPs from human DNA

Amplicons range from 99bp to 500bp.  We have used an appropriate Qiagen kit for extraction.

1. I do not have access to a spectrophotometer to determine DNA concentration, I have seen Picogreen recommended, however this still requires a spec.  Can I just use a DNA ladder to estimate concentration or will this simply not be accurate enough?  Otherwise we'd have to buy a spec + accessories + picogreen + reagents, etc...

2. After performing the PCR off my DNA template, can I use an ethanol precipitation to clean it up before running the Beckman Coulter DTCS kit (BC suggests the qiagen PCR clean-up kit)?

3. I know the CEQ 8000 can do SNP analysis, anyone have experience with this?

Any help would be greatly appreciated, as I do not have any co-workers or friends that have experience with this machine either!  It's brand new to all of us.

[mdfenko]

we have a ceq 8000.

1. an accurate determination is desired. we've seen that it is best to use ~100 fmoles of template for best results, more may clog the capillaries, less may not yield enough product to get accurate results (despite what b-c says about optimum amount of template).
since you will be using pcr products as template (?), you can safely go over the limit by a small percentage, so you may be able to get away with comparing to the ladder.

2. the reason to use a pcr cleanup, rather than ethanol precipitation, is to eliminate the original template dna as well as the reactants. if you don't want to use the qiagen (or similar) spin column pcr cleanup then you'll have to clean the pcr product(s) on a gel. then you can ethanol precipitate it after extracting from the gel.

3. snp analysis on the ceq is performed by fragment analysis. the kit is different (you don't use dtcs). we've never performed snp analysis with this machine (we do perform fragment analysis).

[contactsports]

mdfenko, on 06 June 2012 - 11:56 AM, said:

we have a ceq 8000.

1. an accurate determination is desired. we've seen that it is best to use ~100 fmoles of template for best results, more may clog the capillaries, less may not yield enough product to get accurate results (despite what b-c says about optimum amount of template).
since you will be using pcr products as template (?), you can safely go over the limit by a small percentage, so you may be able to get away with comparing to the ladder.

2. the reason to use a pcr cleanup, rather than ethanol precipitation, is to eliminate the original template dna as well as the reactants. if you don't want to use the qiagen (or similar) spin column pcr cleanup then you'll have to clean the pcr product(s) on a gel. then you can ethanol precipitate it after extracting from the gel.

3. snp analysis on the ceq is performed by fragment analysis. the kit is different (you don't use dtcs). we've never performed snp analysis with this machine (we do perform fragment analysis).

Thank you for responding so quickly!  I figured the ethanol precipitation wouldn't be enough.  Have you ever tried the Diffinity Rapidtip pcr purification method (http://www.diffinity...ts_rapidtip.asp)?

I ran a trial run throught the machine and got the puc18(?) control to work (trust me, that surprised me), so I am hopeful that I can get some results with the help you have provided me.  I'll probably be back with more questions once I have the proper template DNA for the machine.

[mdfenko]

contactsports, on 06 June 2012 - 01:16 PM, said:

Have you ever tried the Diffinity Rapidtip pcr purification method (http://www.diffinity...ts_rapidtip.asp)?

i haven't tried them but they look promising. if you try them let us know how well they work.

Quote

I ran a trial run through the machine and got the puc18(?) control to work (trust me, that surprised me), so I am hopeful that I can get some results with the help you have provided me.  I'll probably be back with more questions once I have the proper template DNA for the machine.

shouldn't be a surprise, it's a standard that comes with the kit with well defined template and primer. i would be more surprised if it didn't work. then either the kit is bad or your pipetting is.

Edited by mdfenko, 07 June 2012 - 11:39 AM.