problem rt pcr long gene - (Jun/14/2007 )
hi! i'm new in this field.currently i'm trying to get full length cdna for 2 genes which beta-glucosidase n endoglucanase,both from aspergillus niger.i'm using rna template of a.niger from 4days culture in mendels medium induced with filter paper.my beta glucosidase gene size is 2.6kbp,endoglucanase is 1.3kbp.i'm using access rt pcr one step kit from promega.i never got my product,my gel always blank,no band at all.i've done positive and negative control shows that the kit is working and no cotamination occur.i've tried change the temperature still didn't get any band.can anyone give any suggestion or comment?i've done all this work for 4 month.i'm really sad...
You can always clone a long pcr.. but its not a good idea..
design different primers from inside the gene... suppose you have 3 kb DNA to pcr.. then u should design 3 pair of pcr primers to aplify 1kb each..
i always do the nested pcr to check the specificity...
other thing is .. that.. we don't know about your control gene??? are these provided by the promega? how long it is? n how long is your cycle?
you might have reaction cycle of short time.. company provided controls are generally not big DNA...
so what could be happening that... short control DNA would be amplifying only... but this cycle may not be enough for your long sample DNA.. therefore you are getting control DNA band... but not of your sample..
you can download full PCR protocols-an ebook Methods in Molecular Biology to learn about the other approaches..or troubleshootings..
thanks "Laboratory HelpDesk" for your answer.
yes,promega kit was supplied with positive control gene (~350bp). i did 45 cycles as recomended by the company.i also didn't know which part didn't work,reverse transcription or amplification.my 1.3kbp gene also fail to get.i've tried 2 annealing temperature for this gene :
1)59.1 degree (average Tm for both primers)
2)56.1 degree (lower the Tm from average)
I don't know where to start to optimize. .temperature or reaction mixture?is it any possibility that my RNA didn't content my gene?maybe my culture condition didn't express the gene out,so my rna didn't have my gene.i use TRIZOL method to extract my rna and use total rna as my rt-pcr template.from the company manual,it's ok to use total rna.
please help me...tq
Had you tried PCR using the RT reaction by utilizing different primers specific to the normal house keeping genes of aspergillus niger? If that worked, then it's more likely that your RT product is ok.
A fast way to troubleshoot for me would be to lower the temperature to less 5 degree Celcius or Tm at 50 degree C. Since you have a clean background with no product, it's worth a try.
tq 'I love MSGs!'. i never tried different primers specific to the normal house keeping genes of aspergillus niger. yes,i will try lower down the temperature.tq