i am purifying cycle sequencing PCR reaction. i have tried multiple methods given in BDT manual (provided with the instrument) .everytime i used 96 well reaction plate for this purpose. 7 wells are used to purify sample while 8th well contain control . i have never seen pellet during this purification process. when i run this purified reaction on genetic analyzer i got results in just 3 wells, control results were fine quality, 2 wells were giving just primer sequence, other 2 wells didn't gave any results not even primer sequence. everytime i get this kind of results. i tried to manipulate the protocol by increasing the value of g ( for centrifugation) e.g from 1870g to 2780g. but all in vain.how can i overcome this problem? do anyone have any method which can give 100 % results. either this problem is related with my purification protocol or some other reason?
Hi Nargis
before trying to purify your PCR, the question is did your PCR succeed?
maybe you should try to put the reaction on BET gel just to be sure to get a band, and therefore the reaction works. just to know, quantity of your amplicon is too small to get pellet and see what ever.
stay safe
fred
You would not expect to see a visible pellet from a cleaned PCR reaction. Also, not all sequencing reactions work. They can fail due to low quantity or poor quality of starting template, inefficient primer choice, etc.
I'm assuming that you are sending out the cleaned sequencing reactions for Sanger?
Check the cost of having the clean-up done at the sequencing center. Given the reagent cost and time you would spend doing the clean-up step, it might be cheaper to have it done elsewhere.
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