Cycle sequencing (Big Dye) - (Apr/20/2011 )
Maddie on Fri Apr 22 18:20:23 2011 said:
I agree with phage. A PCR with too little or no DNA would look like that. Another possibility is that you used too much DNA. 600ng from PCR product is far too much. I attach the manual of the kit, see page 2-6. For PCR product, it doesn't go higher than 30 ng.
How did you quantify your DNA?
thanks for the manual kit maddie. But i did the cycle sequencing manually. Is it ok if i use 30ng? i quantify using spectrophotometer. if my concentration is higher, than i dilute them using m1v1=m2v2 formula.
On the goldilocks subject of too little or too much DNA,
Maybe this might be useful.
I use 1ul of Bigdye in a 10ul reaction for sequencing plasmid DNA. 0.5ul bigdye for PCR product
The amount of DNA used is 25ng * kb (DNA size)
I am new in the field and saw this posts, I was planning to sequence my PCR product with normal sequencing and was told it has to be pyrosequencing. I can't understand why? So you are amplifying your products with bigdye? I wouldn't like to spend so much money in biotinylated primers! What would be the advantage of pyrosequencing? Thanks!!