Hello all,
We do absolute quantification using plasmid as standards which is used in a ten fold dilution. We use a new ABI 7500 machine which has two plates one for the tubes and the other for the plate.
We mainly use tubes from Bio - Rad. Initially we did notice much changes but now 7 months of use of the machine, we started noticing that the synthetic material around the placement which holds the tubes is loose and hence sometimes we see that the tubes do not stay flat on the machine. The tubes end up with a smiling angle. The first and last tubes being the ones that have the rise.
There was variation in the reads like : Replicates stopped being replicates, Low Ct value and Melt curve 2 bands even though running the pcr product on a 2.5% gel did not show any other band or excess of primer.
We did the same assay using the same plasmids and reagents and this time we used the 96-well plates...........And we got the replicates back. Everything was good. Could this mean that the tube bending causes so much difference......
Has anyone here had a problem like this, If so how did you solve it??? Most bio - rad and other machines have slots for the tube to sit in and hence there is less variation. but ABI 7500 the tubes are hanging and once inside the block comes up, hence if the tubes bend.
Thekid..
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