I want to amplify a gene from PPV virus. The gene size is 1350 bp but after pcr i see very faint band of this size and intense band 1/2 size of this band [slightly bigger than 700bp] and one more band at 400 bp.
I tried altering pcr conditions [I am using pfu enzyme] but not success.
I gel extracted the 1350 bp band and did pcr again and got same result.
then I saw the purified pcr still has about 30-40% intense 700bp band.
I sliced only the 1350 bp band but i dont know why the 700bp still persists in the purified sample.
Because of many problems I changed the primers and order new primers that are expected to amplify 1530bp band
but here again the pcr failed i got pcr amplification of 420 and 380 bp.
I used Viralgene spin ViralDNA/RNA isolation kit. I havent treated with RNase - could this be the reason ???
could the 1/2 size Dna be single strand of 1350 bp product ??
Hi, I am very new to virus field. I want to isolate genomic DNA of virus [parvovirus].
My friends told me there re 2 typed of protocols - 1. Involves cell lysis [freeze thaw] the use the supernatent for DNA isolation
2. Directly pellet the cultured cells and use for DNA isolation
I have genomic isolation kit for bacteria. Is it necessary for me to order new kit for Virus DNA isolation ?
Which one of the above protocol should be used ? I need the Genomic DNA for PCR ?
Thank you for your time.
I have read many studies genrerally inactivate the serum used in assays - inactivation at 56 degree for 30 min.
1. Why this is done ?
2. Is this inactivation done only for specific experiments like - Invitro inhibition, ELISA etc ?
I am doing elisa to measure the mouse IgG.
I use mouse sera 1:25 dilution; second antibody at 1:2000 dilution; blocking is with 5% BSA 37 deg for 2 hr. Detection is with TMB substrate
The problem is - I am never able to get negative control [before vaccination] samples values below 0.2. I tried different dilutions of sera and secondary antibody but If i use more dilutions - the positive sera readings also drop.
I tried FBS, Skim powder and BSA - all give similar readings -
I also tried sera from - new mice - 0 day - but still same thing happens
I am wondering If I should change the seconday Ab ? Is it possible that BSA still allows my current secondary Ab to diffuse througn ?