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zhongmindai's Content

There have been 6 items by zhongmindai (Search limited from 24-September 19)


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#109204 18s as endogenous control for Oligo dT RT

Posted by zhongmindai on 08 May 2011 - 04:49 AM in PCR, RT-PCR and Real-Time PCR

HEllo everybody,
I have a lot of experience in qRTPCR but I need some back up voice. Recently I moved to a new lab where Oligo dT is used in the RT step. But 18s is used as endogenous control. Its SYBR green PCR and there is no DNAse treatment. My take is that a. you cannot use 18s as endogenous control because you are using Oligo dT and b. the amplification you see is probably DNA.
Am I right or wrong, do I have much much more to learn about qRTpCR or is there a "mRNA" population for the 18s rRNA that can be specifically amplified using the "right primers"??
Please give your opinion.


Some rRNAs may be polyadenylated for their decay, so it is possible to use Oligo-dT in RT to reverse transcribe these rRNAs. However, I am not sure if these rRNAs could be used as endogenous control as themselves may be differentially regulated.



#95892 Loss of bands at higher Tm in presence of DMSO...

Posted by zhongmindai on 25 December 2010 - 01:21 AM in PCR, RT-PCR and Real-Time PCR

Hi, you misunderstand my meaning. If you performed PCR at annealing temperature of 66oC with 5% DMSO, it is likely that you performed PCR at 69oC with normal PCR buffer.

Yes, but if DMSO lowers the melting temperature, AND my PCR works at lower temperatures, then why do my bands disappear?? I.e. According to your calculation, 5% DMSO would lower temperature by 6oC. Therefore, 66oC with 5% DMSO becomes like PCR performed at 60oC and 64 becomes like 58. But I have bands at 60oC and 58 without DMSO, but NOT at 64 and 66oC WITH DMSO.

I hope this is making sense to somebody...?




Hi,

I am trying to utilise DMSO in order to perform PCR to detect methylation status (http://jmd.amjpathol...bstract/9/5/574). This protocol depends on the fact that methylated and unmethylated DNA show a different sensitivity to the amount of DMSO in the PCR reaction- methylated DNA needs more DMSO in the reaction to show bands.

I have performed a gradient from 56-66oC. (The annealing temp for my primers should be 57-58oC.) At each temperature, I used a master mix with 0%, 2% and 5% DMSO. My hope was to identify a temperature at which the PCR only worked with the addition of DMSO.

However, my experiments are producing very strange results. The addition of DMSO seems to be hindering the PCR reaction. At 56-60oC, the bands with increasing DMSO are fainter. And at 64-66oC, the bands even disappear! I have attached a picture of my results.

I have read numerous posts saying that DMSO really helps annealing and therefore improves specificity and efficiency of PCR. Indeed, the protocol from the paper that I am trying to follow relies on this fact. So why am I getting this result? I really don't understand. Can anyone offer an explanation?

Thanks so much!

DMSO will decrease the melting temperature at about 0.6oC per 1% (v/v). It improves PCR specificity by preventing nonspecific primer-template annealing, as it increase the binding strigency. DMSO improves the efficiency of GC-rich PCR as it helps template denaturation.

It likes that the increased DMSO concentration prevent the primer-template annealing at higher temperature in your experiment.




#94837 Loss of bands at higher Tm in presence of DMSO...

Posted by zhongmindai on 14 December 2010 - 05:12 AM in PCR, RT-PCR and Real-Time PCR

Hi,

I am trying to utilise DMSO in order to perform PCR to detect methylation status (http://jmd.amjpathol...bstract/9/5/574). This protocol depends on the fact that methylated and unmethylated DNA show a different sensitivity to the amount of DMSO in the PCR reaction- methylated DNA needs more DMSO in the reaction to show bands.

I have performed a gradient from 56-66oC. (The annealing temp for my primers should be 57-58oC.) At each temperature, I used a master mix with 0%, 2% and 5% DMSO. My hope was to identify a temperature at which the PCR only worked with the addition of DMSO.

However, my experiments are producing very strange results. The addition of DMSO seems to be hindering the PCR reaction. At 56-60oC, the bands with increasing DMSO are fainter. And at 64-66oC, the bands even disappear! I have attached a picture of my results.

I have read numerous posts saying that DMSO really helps annealing and therefore improves specificity and efficiency of PCR. Indeed, the protocol from the paper that I am trying to follow relies on this fact. So why am I getting this result? I really don't understand. Can anyone offer an explanation?

Thanks so much!

DMSO will decrease the melting temperature at about 0.6oC per 1% (v/v). It improves PCR specificity by preventing nonspecific primer-template annealing, as it increase the binding strigency. DMSO improves the efficiency of GC-rich PCR as it helps template denaturation.

It likes that the increased DMSO concentration prevent the primer-template annealing at higher temperature in your experiment.



#71200 DNase 1 treratment of RNA

Posted by zhongmindai on 16 May 2010 - 09:49 PM in PCR, RT-PCR and Real-Time PCR

Dear zhongmindai
I did not add RNase inhibitor during the dnase 1 treatment of RNA. I shall try adding the rnasin inhibitor to the RNA prep when DNase 1 tretament is given. I have a few doubts
1. Is the RNase inhibitor heat stable?
2. Will addition of RNase inhibitor affect the Reverse transcription step of the RT-PCR.
3. Is it necessary to add rnase inhibitor during the RT-PCR step.
pls help


Most RNase Inhibitor is not heat stable (a company said their kind of RNase inhibitor-like products can work at higher temperature such as 50 oC, but I forget its commercial name).
Addition of Rnase Inhibitor prevent the RNA from degradation during reverse transcription. We always add the RNase Inhibitor in our reverse transcription reactions. RNase inhibitor is necessary unless all your reaction mixtures are rnase free.



#71089 BstUI digestion

Posted by zhongmindai on 16 May 2010 - 12:24 AM in Molecular Cloning

The BstUI from Fermentas is actually Bsh1236I (BstUI), it means Bsh1236I (Fermentas) and BstUI (NEB)recognize and cut the same sequences, but the the two enzymes are from different strains of Bacillus, so they fever different temperature conditions. 37 oC is recommended for Bsh1236I (BstUI).



#71088 DNase 1 treratment of RNA

Posted by zhongmindai on 15 May 2010 - 11:50 PM in PCR, RT-PCR and Real-Time PCR

I agree with pDNA that RNase such as RNaseA and RNaseT1 are very stable, and they cut RNA very quickly. Have you used the RNase Inhibitor (or RNaseIn or RNAguard) in your experiment? Without protection, even chase amount of conteminated RNases will cause your RNA degradation. Make sure your RNA is pure enough, and make sure the DNase I and EDTA you used is certified to be RNase free. Finally, always add the RNase Inhibitor or something like that in the reaction mixture to protect your RNA.




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