Few days before I had asked the the same question and this problem not resolved yet. If any one want to check that discussion can go my profile check 1st question and discussion and then come into this it will give more clarity of my PCR problem, and please give me possible reason of this problem and give solution that how I will overcome this problem?
The problem of getting band in every samples is continued. This time I have designed new pair of primer sets, One primer set is on flanking region of gRNA on T-DNA and another primer set I have designed on the Cas9 region on T-DNA. Before ordering this primers I have blast both the primer sets and those are not binding in any genomic DNA region of any organism. Then when it came ,For dilution of Primer mother stock I have used fresh autoclaved water from other lab and pipettes also I have used from another lab.
Before doing PCR I have Isolated fresh genomic DNA and all reagents I have prepared in fresh. Then I have put PCR but again I have got bands in every sample including Wild type and Water control also. after this experiment I thought primer might contaminated or PCR pre-mix has contaminated,
so next PCR I did on another lab and only new aliquot of Primer and new isolated genomic DNA, and this time one thing I also Included that I have used genomic DNA of Chickpea, Medicago and Peanut with two water control but again I got bands in all of my 5 samples with 2 rice wild type , out of 2water control one had very very faint band other not and in chickpea and peanut I got band not in Medicago.
The primer I had used
FP Primer Tm 64
RP primer Tm 65
I used annealing temperature 60, and amplicon size is 580bp
So, now What I will do?
For more clarification of my problem our lab have used more than 10 pairs of primer and all the time we are getting same result. Frequently one thing happen in our lab is that primers are getting contaminated but sometimes the primers are ok and we didn't get any band in wild type and water control but band in every samples then we go for sequencing with or without cloning in PJET vector, then also we noticed that the gRNA which should be present on that clone that gRNA is not mine that gRNA from my another lab mates cloned gRNA, that clone He or she still didn't use for regenerating plants, It means the contamination of his or her cloned vector in my PCR reaction but in which stage this contamination is coming we can't understand -
1) During isolation of genomic DNA?
2) during PCR?
3) During PCR elution?
or 3) During sequencing?
or 4) those all primers no are not good for genotyping?
Now What should I do?
Contamination happens. Your goal is to get the PCR working, not to track down the source of the contamination.
Do you use the same pipettes for loading gels that you use for setting up PCR? That's a common source of contamination.
Do you put amplified DNA on the same bench/same area that you use to extract DNA or set up PCR? Also a common cause of contamination.
Get a different primer for the T-DNA & try again.
Thank you so much,
Yes its not to track down the source of contamination but in which stage this contamination happen, so I will avoid those things to get Good PCR.
No I didn't use same pipettes for loading and setting up PCT, For loading we kept separate pipettes.
yes we I use same bench for putting PCR where I isolated genomic DNA. Ok I will avoid this.
Ok I will design new Primer will try again.
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