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ashu2007

Member Since 16 May 2012
Offline Last Active Yesterday, 10:02 PM

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In Topic: High fidelity PCR trouble shooting

Yesterday, 09:44 PM

uday, on 23 May 2013 - 06:59 PM, said:

I want to amplify a 1.8kb gene using error prone PCR for subsequent cloning purpose. My primers works well with normal tag polymerase (Fermentas Dreamtaq Green PCR master mix) and gave me the desired product. However, when I use the same primers and template with high fidelity tag polymerase (Platinum Tag DNA polymerase high fidelity - Invitrogen), I can't get any amplified DNA. What was the wrong? what would need to be changed? I am bit confused Posted Image

Hi Uday,

Because of high fidelity, might be this enzyme is slow. You can try to use mix of Taq and this high fidelity enzyme. Mix them in different ratio (1:1, 2:1). I hope this will work.

In Topic: Inconsistent sample quality in quantitative real-time PCR - What could be the pr

29 October 2012 - 08:47 AM

I had the same problem but now it works fine for me. I think you don't need to make mRNA first before making cDNA. I use my home made recipe for this. I use total RNA and convert that into cDNA. Titrate your RNA with primer and water.

Heat it at 65 degree for 10 minutes, then cool it on ice for 5-6 min, then mix your RT enzyme, buffer and Mgcl2 mix it properly and keep it at 42 degree for 1 hr.

Then do a normal PCR to confirm it. Use the same condition as PCR but this time include SYBR green.

Suggestion: run those product (which you get from qRT-PCR) on a gel, which will give you an idea about your product. Do pipetting as less as possible, because most of the time pipetting error creates problem in this experiment.

In Topic: Real time PCR sudenly not working

11 July 2012 - 08:50 AM

eldem, on 11 July 2012 - 07:52 AM, said:

Hello to all,

I am new to real time PCR, but I have done some successful experiments with Homemade protocol. I use AB7500 standart machine. A far as I know other people that use the same machine in the same time don t have problems. My problem is that one day I have normal Curves with Ct s around 28 and the next day I have no amplification at any well of my 96 well plate.
I have checked my aliquots (primers, buffer, enzyme, dNTPs, MgCl₂ ) and they work OK.I can not imagine what goes wrong some times.
I would appreciate some help
Thanks.

Hi,

Have you run the product which you got after your qRT-PCR. First run that in a gel which will confirm that you are getting product or not. Even I am using home made protocol for qRT PCR. Sometimes SYBR GREEN creates problem. Make a stock of 100X dye, whenever you want to use, dilute it and use it after use throw that away.

In Topic: Qiagen PCR Array Reagents?

28 June 2012 - 04:31 PM

I made mastermix by myself and added SYBR Green in that mixture. It is working fine for me.

The disadvantage of this is, first you need to Optimize conditions using cDNA RT PCR then only you can go for qRT PCR.

In Topic: Application of RNase

28 June 2012 - 07:32 AM

Nephrite, on 30 May 2012 - 11:55 AM, said:

ashu2007, on 30 May 2012 - 07:48 AM, said:

Nephrite, on 30 May 2012 - 07:34 AM, said:

Hello.

My RNAs are extracted from 10 paraffin embeded tissue sections/sample. I used the Qiagen kit with columns. The amount of RNA in all samples is app. 40-50 ng/ul in 20 ul volume.

A reviewer recommended two different approaches to check if there is contaminating gDNA. One of the approaches is to treat my RNA samples with RNase and then to measure for gDNA.

I intend to measure it before and afer treatment, but I don`t know how to deactivate the enzyme. In the instruction manual (Fermentas) they say that heating of samples will not deactivate the RNase and they suggest chloroform/phenol extraction.
However, my sample is RNA predominantly, in little amount and small volume.

Do I really have to perform this extraction step (which I don`t know how in this small volume)?

Can I try to denature the enzyme with some low concentration of......SDS or something? Will it interfere with the NK?

Do I really should deactivate this enzyme just to perfom 10 min measurement? I will not use these samples after that.

Thank you.

Even I faced the same problem whenever I used kit I was getting around 100-400ng/ul concentration of RNA and my RNA was degrading because of RNase from some sources. Now I started using Normal manual method and I made solutions in DEPC treated water. Now there is no problem.

I will suggest you go for the phenol choloroform extraction, because this is the best way to get rid off any possible RNase contamination if you have in your RNA and it will concentrate your RNA. With the manual method Now I am getting 1-2ug/uL concentration of RNA.

Thank you for your kind and quick reply but I actually want to destroy all RNA in my RNA samples and to measure the gDNA incontaminants :-) The meaning I guess is that it will increase the concentration of gDNA if the RNA has been destroyed. I just wonder do I have to deactivate the enzyme after that :-)

I think RNA can be destroyed by RNAse with in 10 minutes and after that keep it for 10 minutes at 65.C so that whole RNase will be deactivated.

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