No electropherogram peak - (Dec/06/2006 )
I did sequencing of the 16s rRNA of my bacterial DNA samples. However, the resultsfor my 24 samples were blank as I didn’t see any electropherogram. It is my second sequencing and I did the same procedure as my successful sequencing before. What could be the possible reasons here?
u need to check these
1. did u get a primer peak after 25-30 min of run.
2. was there a signal but very weak.
if u have not got the primer peak then there cud be either no extension of ur insert. or u would have lost the sample during the post seq rxn clean up (pptn and 70% washes etc). lack of extension cud be also bcoz of the primer problem ( u need to check if its working fine by doing a normal PCR rxn).
if there is a very weak signal then u need to increase the quantity of the sample loading (u need not do a second seq rxn but try to run the same rxn the 2nd time with more sample (in MegaBace generally increasing the volts or the time for load helps to get in more sample).