27 replies to this topic

[umam]

Hi,

The NTC gives me a peak almost every time, I set up a reaction. Currently I am using Roche 480 and analyzing expression of my target gene compared to the housekeeper i.e actin. One other problem is that in my untreated samples (I work with mosquito larvae and expose them to bacteria and use real time PCR to check presence and no. of bacteria) I am getting a low Ct value for my bacterial specific gene, but I am confused as to why would I see any presence of bacteria in my control samples? Also, my target bacterial gene is very specific and should come up positive only if my bacterium is present in the mosquito larvae. Also, is actin expression affected by anything? AS in since actin is my housekeeper gene, should i not be getting the same levels of actin present in day 1 of my treatment and on day3 an day 9? Does it vary with size of the larvae? Is there anyway I can ensure that I have the same amount of actin across all my samples?
Please help..
Thanks,
-Uma

[oxygene8]

Quote

I am also having some issue with, contaminations but it is extremely bizarre, it is not in all wells! When I repeat my PCR I do not always get the same positives (this a PCR for genotyping) and sometimes I get a positive band in my blank. I have tried changing all my reagents (including my water), cleaning my pipettes, even using somebody else's pipettes with their tips. I am not sure what to do anymore. I do not have many choices for my primer sets as the transgene is very similar to the endogenous gene. Any suggestions?

I faced this problem before and the culprit were the PCR tubes. Especially if u use strips without individual caps (or worse, a whole plate with the film covering) this could be a problem since samples in one strip are open till reagents and samples are added in all other wells and closed with the common strip cap or film. And I had this other experience where the tubes had faint cracks in them, not seen with the naked eye, but do make sure ur products are the same volume after the run to ensure u havent used a cracked tube. This could be a possible source of contamination from one run to another.

[Adrian K]

Regarding my experience about the false positive in NTC which happened 2 weeks ago, I changed my PCR buffer and MgCl2. I did not face any false positive after that. Hope this helps.

[uvbox]

uvbox, on Sep 5 2009, 12:31 PM, said:

Two of my -RT are getting faint bands, could it be due to gel loading problems, where i had some +RT not going into the well and it might have travelled to my -RT well beside it??

I think i might also have lots of DNA as my samples are from fresh human blood and i've extracted RNA from it. Would it help if i bump up my DNase I enzyme treatment? e.g. use 3ul instead of 2 ul etc

Any help much appreciated!

Edited by uvbox, 04 September 2009 - 04:33 PM.

[UBClabbie]

the most common source of contamination in my qPCR reactions have been from the air. aerosols do exist and can cause contamination.
desperate measures call for a hood! use a laminar flow hood or even better a biological safety cabinet to set up your reactions. if you use a bsc you can sterile it before hand by turning on the UV and the fan for 20 minutes prior to setting up your reaction . just like when you're performing cell culture experiments, limit the amount of items that pass above your reagents, that is, keep from reach your arm overhead of anything open. this can lead to contamination. pipette things on an angle. close container immediately after use and don't open them until you are immediately going to use them.

Good luck! wait a couple days between reaction setup to limit contamination and tell your lab mates to keep any materials, including their gloves away from the areas you are going to use for setup. I freak out when people bring their gloves from the gel room into the qPCR room.

[gaurav amit prakash]

hey

it is seen that sometime the lab platform where u do all reactions is not able to be free of contamination so try to carry uor work in a different lab or place away from dust etc. i think it will work definitely. also tell us that is uor required band is coming or not.???

[muntasir]

It is better to clean your pipettes, working area with 0.1 M HCl before and after PCR. It removes contamination.

[sciencelover]

If you are using the 8-strip PCR tubes, their is a lot of cross contamination issues because of the lid. Try using these [url="[url]http://www.alkaliscientific.com/en/pcr-test-tube/316-pcr-tube-8-strip.html"]PCR[/url] tubes[/url] with individually attached caps. They prevent splashing the solution around causing cross-contamination. Plus, you can cut them.

[frna11]

I understand most of the advice given, but am not sure why you recommend making the master mix away from the normal PCR bench area. Is this because in that area there will be a lot of different plasmids/DNA samples being used for PCR? In that case wouldn't anything you do there be as liable to contamination as working at your bench? For that matter.. Why is it recommended to use a seperate area for PCR? If it is not cleaned regularly it is seems as likely to contain plasmids/aerosols as your regular bench. Am I missing something? Or are you just meant to clean it

[Zaffer Zargar_28259]

Dear all hi..
m i first year of my phd  and right now i am facing lot of problems...particularly in the real time pcr..the problem is that i am getting "SPECIFIC" amplification also in NTC ... m target band is around 180 bp which i am seeing in the samples but also in NTC ..m doing real time PCR for checking promoter occupancy of some proteins using CHIP... i have tried everything i could do to get rid of this problem.. cleaned pipettes, changed gloves, changed water. autoclaved Sigma water and then kept it in UV..kept pipettes in UV hood ..changed primer stocks, changed syber green .. i have done everything me and my boss could think of. i tried doing the PCR set up in hood...even i  gave my samples for some other persons in lab even they got the same porblems... both NTC and samples are showing amplification at the same Ct value...looking forward for help by u people
Bye...

[Trof]

Did you sequence the NTC product to really know it's specific? Was it ever negative, is this a newly designed or validated assay? Other genes you run are fine? What Cts are your NTC/sample, is this Ct comparable to the other genes Cts or higher (or different from what you would expect given the hypotesis)?

[Zaffer Zargar_28259]

I have not tried sequencing it..the Cts are around 30 -35 for both samples and NTC ..i have done real time for GAPDH before for that Ct was around 24 and there was nothing in NTC at that time but that i was doing from cDNA as template but in this case m using DNA eluted after CHIP assay..These primers have been used by other people before without any problem. NTC was negative but only once .

[muntasir]

Hey Zaffer ... Have you solved the issue? If yes, please let us know, how?