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dear pcrman.
thanks for your explanations. however, for BDNF alone, i checked and found out that there are more than 700 SNPs reported for this gene (though only around 20 on the active gene site if im not mistaken). So instead of doing 6 times of genotyping for the particular 6 SNPs i want to look into, can i just proceed and do one genotyping for the whole gene sequencing for that particular gene? As per mentioned earlier the active gene site for BDNF is 247AA-long.
thanks.
Posts I've Made
In Topic: Left my media at room temperature for 48 hours; okay to use?
04 July 2012 - 03:19 AM
I guess you can still use them, if your room-temperature is around 18-20"C, as in an air-conditioned lab... 
of course, do bear in mind that some components may be slightly denatured. so i guess maybe you can increase or even double up the volume for culturing your cells... eg. instead of 3-5ml for a T25 flask, put maybe 6-10ml...
of course, do bear in mind that some components may be slightly denatured. so i guess maybe you can increase or even double up the volume for culturing your cells... eg. instead of 3-5ml for a T25 flask, put maybe 6-10ml...
In Topic: Free Cell Culture Handbook
04 July 2012 - 03:05 AM
thanks for sharing! have anyone of you tried to get your free copy of various literature of Sigma-Aldrich? You can do so through http://www.sigmaaldr...re-request.html~ Do share with other research fellow friends of yours...
Take care~
Take care~
In Topic: Problem of MTT Solvent (10% SDS in 0.01M HCl) for dissolving formazan crystals
01 July 2012 - 05:02 AM
Hi Seng and EvilTwin.
My protocols are kinda similar to Seng's, to a certain extent.
Seng's protocols:
1) Plate 100ul cell suspension.
2) Add 100ul extract directly without discarding the spent media (to reduce variation).
3) Add 20ul 5mg/ml MTT solution and incubate for 4h.
4) Add 50ul 10% SDS/0.01M HCl
5) Incubate in 5% CO2 incubator at 37oC for 1h before measuring at 590nm.
My protocols:
1) Plate 180ul cell suspension, and incubate for 24h (for cells to adhere to the microplate walls).
2) Add 20ul extract directly, and incubate for 24, 48 or 72h (treatment time).
3) Add 20ul 5mg/ml MTT solution and incubate at 37"C, in dark, for 3h.
4) Remove gently all or most of the media in the wells, and add 200ul DMSO.
5) Agitate the microplate for 15min via orbital shaker or microplate shaker, and measuring ODs at 554nm (with reference at 690nm) within 1h of adding DMSO.
Maybe you can try using DMSO as the solvent? I came across few papers stating that isopropanol may not be a very effective solvent to dissolve formazan salt. No idea for SDS though, cause never heard of it before...
Anyway, hope it helps. Take care~
Tony.
My protocols are kinda similar to Seng's, to a certain extent.
Seng's protocols:
1) Plate 100ul cell suspension.
2) Add 100ul extract directly without discarding the spent media (to reduce variation).
3) Add 20ul 5mg/ml MTT solution and incubate for 4h.
4) Add 50ul 10% SDS/0.01M HCl
5) Incubate in 5% CO2 incubator at 37oC for 1h before measuring at 590nm.
My protocols:
1) Plate 180ul cell suspension, and incubate for 24h (for cells to adhere to the microplate walls).
2) Add 20ul extract directly, and incubate for 24, 48 or 72h (treatment time).
3) Add 20ul 5mg/ml MTT solution and incubate at 37"C, in dark, for 3h.
4) Remove gently all or most of the media in the wells, and add 200ul DMSO.
5) Agitate the microplate for 15min via orbital shaker or microplate shaker, and measuring ODs at 554nm (with reference at 690nm) within 1h of adding DMSO.
Maybe you can try using DMSO as the solvent?
I came across few papers stating that isopropanol may not be a very effective solvent to dissolve formazan salt. No idea for SDS though, cause never heard of it before...Anyway, hope it helps.
Take care~Tony.
