November 4, 2009 at 4:28 am #12200jasminaParticipant
I used specific gene knockout mouse for experiment, but these days, my results are horrible, so i want to check wether this mouse is knockout mouse or not. I have done genotyping experiment 2 times, but i could not see any bands after electrophoresis. I think may be my samples extraction methods has some problem, can you indicate the incorrect steps for me? or can you provide me a new protocol for samples preparation?
thank you very much!
following is my protocl for samples preparation, i used mouse toe for experiment.
1 add lysis buffer(200ul) and proteinase K 2ul in tissue
2 overnight in heat block at 55℃, 600rpm
3 add phenol(PH7.5-8.0)—Phenol:sample =1:1
4 mix gently for 5-7min with hand
5 centrifuge for 5min at 12,000rpm, 4℃
6 collect upper phase
7 add phenol:chloroform (1:1)
8 mix gently with hand for 5min
9 centrifuge for 5min at 12,000rpm, 4℃
10 collect the upper phase
11 add chloroform (same amount as sample)
12 mix gently for 5min
13 centrifuge for 5min at 12,000rpm, 4℃
14 collect upper phase
15 air-dry for 2hrs
16 store at 4℃—ready for PCR
Genomic DNA lysis buffer
100m M Tri-HCL PH8.0
5m M EDTA
200m M NaCL
November 4, 2009 at 5:15 am #94483JackBeanParticipant
So, you are drying about 200-500ul of water? Why don’t you use ethanol or isopropanol for that?
Otherwise the protocol seems fine for me, but I have actually never worked with animals 😉 Also, I have never used only phenol for the extraction…
Anyway, as are you doing knock-out mouse, shouldn’t you actually NOT get the band? 🙂 Do you use some WT mouse as positive control?
November 7, 2009 at 2:51 am #94613jasminaParticipant
thank you very much for your reply.
at the end of experiment, i get 50ul chloroform containing genomic DNA, and i dried it for 2hrs.
these days, i searched information about my knockout mouse, i found that, compare with w.t mouse, knockout moue loss 42 base, and when i did PCR, i used different primer, so i can get band from knockout mouse if samples is good enough.
today, i did PCR again used dilution samples( 1/100,1/1000), i get clear bands, however i don’t know why dilution can solve this problem, do you have any idea about this ?
November 7, 2009 at 3:14 am #94614JackBeanParticipant
chloroform containing DNA? I thought, that the DNA stays in the water phase always… 😕
Yeah, I have noticed things like that several times as well, for some reason too much DNA can "inhibit" the PCR reaction, probably as there is lot of DNA, which could hybridize and lot of DNA, where can bind the DNA Pol, so it doesn’t work very well.
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