Thanks for your help and answers Bob1. The protease was resuspended with 300ul of FG3 i.e. before the 4 (sample) practice tests and used the remaining portion of FG3 resuspended protease for both test 1 and test 2 also, which I had accidentally left it out on the bench for an hour or so before test 1 at room temperature and returned to 4 d C as soon as I realised. At step 4, yes I did make the FG2/(resuspended) qiagen protease fresh each time and used within an hour of producing.
During the first test that I realised I missed up, the protease/FG2 buffer would have incorrectly been for 500ul (rather than 300ul) of blood based on 24 samples, where I used the following calculation to create the FG2/protease:
24 whole blood samples * 0.5ml = 12ml whole blood (total)
=> FG2 total = 12 * 0.5ml
= 6 ml
plus protease = 12 * 0.005
Although I had not increased the reagents volumes for 500ul of blood in this '1st' test, instead mistakenly followed the standard protocol as listed in the instructions that is set out for only 300ul of blood.
The 2nd (new) repeat test I decided to follow column 1 shown in table 2 (p. 14) of the flexigene kit protocol which uses 100ul of blood, 250 FG1 (@ step 1), 50ul FG2/protease mixture (@ step 4), 100ul of 100% ethanol (@ step 6) which I substituted for isoproponol, 50ul of 70% ethanol (@ step 9) and 100ul of FG3 (@ step 13); this last stepped I even increased the FG3 volume to 500ul total in each microfuge tube, which seemed to help dissolve the DNA in some samples, with most of the samples needing vortexing to disintegrate the DNA. As part of this 2nd test, I followed column 1 of table 3 (p. 15) with 50ul of FG2 and 0.5ul of
I'm not using specialised pippette tips, so there is some retention of blood (5-10ul blood??) remaining on the sides of the discarded pippette tips and as they warm to room temperature, though the fresh pippette tips don't retain any significant volume of the reagents...Perhaps the slightly reduced volume of blood is not important. I'm just trying to think out of the box.
In my last post I was going to leave the samples in the water bath over night, though instead I took out after one hour and then placed into a heat incubator that has a rotating pad which I set for 999 mins (the maximum) and will assess again tomorrow.
To one of the samples with dense (in appearance), stubborn DNA I added 1.5 ml total of FG3 buffer, it perhaps helped to soften and break apart the DNA pellet so it wasn't so dense. I'm not willing to replicate this with other samples in case my FG3 buffer runs out and I need to run test 3, so difficult to know how effective that was at the moment!! lol. I'm back home and will go back again to uni in the morning to re-evaluate the situation, come what may!
Hopefull this makes sense.