For those who don't know what is PCR. Let's me give a brief explanation about this. PCR stands for Polymerase Chain Reaction, a method use in molecular biology to amplify template gene/DNA, and follow by series of downstream processings.
I mentioned in facebook last time, that I had extract RNA from 400 samples, then to cDNA follow by PCR, gel electrophoresis and qPCR. I did my undergraduate research in the field of phytochemistry, i.e. antioxidant from plant sources. Therefore, shifted from biochem works to molecular, genetic and physiology field are great challenge to me. Besides, catch up with all the technical skills, I also have to do a lot of readings myself..Anyway, this is research..Only passion, prolong interest and commitment will drive you to move forward..hehe
This first PCR, and gel electrophoresis was performed 2 weeks ago. Definitely excited, because had a first hands-on experience, and I seldom do molecular lab work in previous Uni...really paiseh..haha
Basically what I did this three months, mostly on trial and error basis, and made myself familiar and get used to all techniques before proceed to other tough works..
Anyway, nothing much can see from this gel image (both are the same, different contrast). Anyway, when discuss with my post-doc, there might be some "significant thing", anyway, we must prove its significant through qPCR. Also, the low intensity in some band means the PCR cycle might not optimised...
Therefore, what I am going to do now: trial and error again, try varies cycles, dfferent magnitude and so on..OMG
By the way, just received an email from graduate office, about election of course rep, and from the email, 1 representative will be elected for each course. This must be funny, as I am going to elect myself and vote myself..Looking forward for the meeting..hehe...
New year coming soon...hope you guys enjoy it!!!
8 comments:
Those are real nice and thick bands.
i miss PCR.... and qPCR...... T.T
All the best CK and
Merry Christmas !!!
haha,hope so..still have long way to go
This image is very nice =)
the dragging one is it havt finish run?
something in the -ve ctrl, hmm..
FYI CK, marc has phobia for any bands or anything in the 'negative control lane'.
**hehe brings back fond memories of FYP**
XD
XD wakaka,
i'm kinda sensitive towards band after that. ish..
hello,
hi marccus & ange: what means by FYI? haha,by the way, those bands observed at the bottom are uncoupled nucleotide.
smearing in some lanes, and unequal density of band size were due to non-optimised amplication,
this problem overcome in the second trial, by modifying annealing temp.
Hello CK,
Congrates that you got success in getting your bands. It ought to be a very exciting moment when you first see your PCR positive results. You reminded me of my days of Post-Graduation, when I used to pray to God each time I run the gel. Well, I would like to tell you about the two dark patches what is quite visible in your gel's photos here. I hope you are using 6X loading dye composed of Bromophenol Blue+ Xylene Cynol. This Xylene Cynol gives bands of about 300-400 basepairs. Consequently, when you locate your bands in this range, they appear with less intensity as it is in overlapping region of xylene cynol. I would like to suggest you not to use this in your loading dye composition. Its purpose there is to increase the density of the fluid so that is settles down at the base of the well. Inspite of this, you can use Glycerol, the purpose is fulfilled with no hindrance of dark patch. Try this remedy, and enjoy your brighter bands.
Marry Christmas..
NP
India
Dear Neelam,
Thanks for your comment and also your support.
You are right, as my post-doc told me,PCR is 5% joy and 95% disappointment,
You are right, I was using 6x loading buffer, and as a result two distint bands observed. I began to use glycerol in subsequent trials,
Thanks for your elaboration, now i know the contributor of these distint bands,
wish you Merry Christmas and Happy New Year 2009.
cheers.
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