fibonacci

i'm planning to do a relative quantification and from what i know, i have to perform standard curve for each genes that i used in qPCR. actually, i have about 25 genes of interest. so, do i really need to perform the standard curve for 25 genes??

actually, i want to do the relative quantification.. for example, i have 5 target genes and i have 1 ref gene..so i want to compare the expression level of these 5 genes at 3 different conditions (samples are from the same organism)..

HI... actually I need some advices from the forum members... i plan to do the qPCR for several gene of interest (GOI)... and i plan to do the relative quantification... from my reading, i have to test the primers efficiency before i start the real assay.. to do the primers efficiency, i have to generate the standard curve for each pair of primers.. from my reading, i found out that normally researcher will use the 10-fold serial dilution.. so, my questions is : 1) can i use 2-fold @ 5-fold serial dilution of my template??? because i have tried 10-fold dilution but the results is not so good..i think because of my GOI is not highly expressed because the Cq value for 100ng (highest concentation) is ~23.. 2) besides that, do i need to run the assay (in the same running process) for the reference gene each time i run the GOI??? (i mean, if i have 5 GOI, so, i need to run it 5 reference gene???)..i asked this because i have several GOI and several samples...so, i cannot fit all the GOI and reference gene in the same running process... really appreciate if someone can help me... thanks..

thanks for the info... i will try first...

hi.. i need some help from the members who has some experience doing the real-time PCR... i'm planning to do the one-step REALTIME-PCR and i have bought the QuantiFast SYBR Green kit, Qiagen.. i have some questions about real-time PCR before i starting the experiment. the questions are :- 1) how to prepare the standard curve in real-time?? what template should i use to generate the standard curve?? DNA(plasmid DNA which contain my cDNA) @ RNA ?? 2) after i designing the primer, i testing it by doing RT-PCR and i successfully amplified the genes of interest with the expected sizes. but, in the gel picture (2% agarose), i found out that there are some smearing under the expected band (means that the size is smaller than the expected sizes). does this smearing will affect when i do my real-time experiment??? 3) in the QuantiFast SYBR Green kit protocol, it stated that the annealing/extension process were combined at 60C for 30 seconds (it also stated that this temperature should also be used for all primer sets with a Tm well below 60C). i have designing the primers with Tm around 55C-57C.. so, which Tm should i use?? 55C-57C or 60C??? hope someone can help me... thanks..

can anyone explain the principles of lipid extraction (chloroform:methanol , Foltch method) ?? thanks.

when i do the plasmid extraction by using published method by Oloni Kotchoni et al. (2003), i didn'y get any band when analyzed by 1% agarose gel...but when i measure by spectrophotometer, the concentration of the 'plasmid' is quite high, ~1000ng/ul and the purity is quite good... but then, i do PCR colony juz to check the insert of my cDNA..but, i still didn't get any band in the gel picture... actually, this is the 2nd time i do the electhroporation..the 1st time i do it by using same method, i was successfully obtain the cDNA...but the 2nd time, i fail to observe the cDNA in my plasmid... so, any suggestion @ advice for me.... p/s - is there any effect after i do the electroporation, i incubate the cell at 37deg C for three hours????the protocol recommended to incubate at least 90 minutes...

does anyone know how many ribosomal RNA yeast have??? usually when i extract the RNA, i only get 2 intact bands (28S & 18S).. BUT somehow, when i do the RNA extraction, i will get 3 bands... there are another band 'upper' than the 28S rRNA...(but it is not genomic DNA because i run the RNA with 1kb ladder and the size of the 'upper band' is around 4.5kb...) does anyone know about the band???? please help me.. when i measure the absorbance of my RNA, usually i will get a good reading of A260/280 which is around 1.8-2.0....but A260/230, usually i will get around 1.40-1.70... how can i get rid of the polysaccharide contamination????? any suggestion???? p/s - i do the RNA extraction by using TRIZOL from Invitrogen....

i juz beginning to contruct my cDNA library... but after i study the protocol (Stratagene kit), i juz wondering how to calculate the titer of my phage??? if someone know about that, can u elaborate more about this... besides that, i still don't know why i should do the mass excision...

my supervisor said that when sequencing EST, we juz sequence it 1 time and juz 1 strand...if i'm not mistaken from 5'-end... can anyone tell me why???? i'm juz curious.... :confused:

i juz finished extract my RNA ... but the problem is when im check the quality of my RNA, it shows that there are lot contamination of polysaccharides(A260/230 = below 1.0).. the A260/280 is ok, i think...around 1.7-1.9... actually i want to construct cDNA library.. so, how can i get a good quality RNA to synthesize cDNA??? my fren juz tell me that i can purify my RNA before synthesize cDNA.. is it right????? and 1 more thing, if my RNA not so good , what happen if i continue to synthesize cDNA????

i think u can use the nebulizer.... actually it can cut the genome in random size using the pressure from the gas...

have anybody here know about 454 pyrosequencing??? is it much better than Sanger method???? :confused: