I am comparing 2 RT-PCR kits by using the same samples, looking at 2 targets. The top gel is one target, size 101bp. Lanes 1-4 are with one kit, where 1-3 are positive samples and 4 is NTC. Lanes 5-8 are the same samples, but tested with kit #2, in the same order, where 5-7 are positive samples and 8 is NTC. The bottom gel is testing a second target, size 204bp.
I can see that with kit #2 (lanes 5-8) I don't have the top band that I see with kit #1 (lanes 1-4). There also seems to be a smear on the top gel using kit#1.
Any ideas why this is so? Should I try to change cycling parameters? If so, should I focus on changing the denaturing step or annealing/extension?
(pic attached)
Were it not for NTC, I would say that this is a template DNA leftovers and that second kit has gDNA removing step. But you see it in the NTC as well. Maybe some kit/PCR components are contaminated? What components exactly does your NTC reaction have? Is it one step RT-PCR?
Anyway just in case throw away the water you are using for PCR, get a fresh one (commercial, nuclease free for PCR).
Hi Oleksandr. The NTC just has the RT-PCR kit reagents and RNase-free water, which the kit included. This is a one-step RT-PCR.
I am re-running the sample as we speak with new water, new kit, and new primer aliquot, just to be certain.
Fingers crossed!
I am also doing different amount of RNA. The first kit is supposed to be more sensitive, so maybe the original RNA amount was too much?
We'll see.
So is the NTC based on the first kit? Did you do it for the second one?
The NTC is on lane 4 for the first kit. This NTC is just reagent from kit 1 plus water. The water I used was the same water that I used for the cocktail, which is from the kit.
The NTC for the second kit is on lane 8. This NTC is reagent from kit 2 plus water. The water I used came from kit 2, and is the same water I used for the cocktail.
Sorry missed the second NTC description. OK, looks like water or other component from kit 1 is contaminated. Does either of kits have the DNA removing step?
Neither one of them do :/
This was a brand new kit that I used, so I'm a little surprise that it may be the water. In any case, I am using a brand new kit with new water to test kit #1. I am also expecting a call from the manufacturing company to see if anyone else has experienced this same problem or if they have any suggestions.
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