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dtae's Content

There have been 35 items by dtae (Search limited from 17-October 18)



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#119979 CO2 Backup system for -80 freezers?

Posted by dtae on 19 September 2011 - 04:05 PM in General Lab Techniques

Hello,

I've been considering CO2 backup systems for a -80 freezer in case of power-outage. Has anyone used such a system? Are they worthwhile?

According to Fisher a 50 lb cylinder of CO2 should last for about six months to a year without being used and keep a freezer at about -60 for about 8 hours in an emergency. Not sure how realistic this actually is.

Thanks,
Dan



#110078 >100% qPCR efficiency

Posted by dtae on 17 May 2011 - 08:45 AM in PCR, RT-PCR and Real-Time PCR

To see whether the >100 E is due to standard curve artifacts you should consider calculating well-specific E values (e.g. with LinRegPCR if your software doesn't support it).

D



#109164 UV safety in cell culture hood

Posted by dtae on 07 May 2011 - 03:19 PM in Tissue and Cell Culture

see

Meechan PJ, Wilson C. 2006. Use of Ultraviolet Lights in Biological Safety Cabinets: A Contrarian View. Applied Biosafety 11(4):222-227.

less seems to be known with confidence about this than I would have thought



#103236 Real-Time PCR as a Microplate Reader

Posted by dtae on 10 March 2011 - 08:39 AM in PCR, RT-PCR and Real-Time PCR

Any updates on how this works for you?



#103226 VWR Real-Time PCR plates for eppendorf Realplex

Posted by dtae on 10 March 2011 - 08:02 AM in PCR Reagents and Equipments

I've used VWR brand plates with strip caps in a Bio-Rad iCycler with good results.



#103225 real time PCR machine

Posted by dtae on 10 March 2011 - 08:00 AM in PCR Reagents and Equipments

While Rotorgene 6000 is probably much better in terms of having lower inter-reaction variability, they are much harder for larger sample sizes since conventional things like multichannel pippets don't work so well with them.



#102899 How & where to prepare qPCR

Posted by dtae on 06 March 2011 - 12:54 PM in PCR, RT-PCR and Real-Time PCR

ivanbio, I think you're incorrect about the roots of separate rooms for PCR having its roots in the 2004 EPA report. I've worked in multiple labs established before 2004 which had separate rooms, positive pressure and other such techniques in an effort to eliminate contamination.

I've personally worked in several labs with very high levels of measures taken to eliminate contamination (separate rooms, positive pressure, frequent UV and bleaching) and labs with little to no such measures. I've had little trouble with contamination and haven't noticed a difference between labs.

Those interested in the topic should also see:
http://www.protocol-...h__1#entry40274



#102898 Pre-PCR and Post-PCR activities in one room

Posted by dtae on 06 March 2011 - 12:39 PM in Molecular Biology

Bassaml7 - out of curiosity, did you end up having any problems with contamination?



#100347 qPCR for telomere length measurement - efficiency issues

Posted by dtae on 11 February 2011 - 09:45 AM in PCR, RT-PCR and Real-Time PCR

The most important thing with efficiences is probably that your control sample(s) and unknown samples have similar efficiencies in both telomere and scg amplicons. As long as you are correcting for efficiences in your calculation method, I don't think having different efficiencies between telomere and scg is a big deal.
I would suggest looking at your efficiencies on a well-by-well basis using LinRegPCR as well as the standard curve method. I bet you'll get substantially different results.

Dan
Department of Anthropology
Northwestern University
www.dtae.net



#98520 qPCR for telomere length measurement - efficiency issues

Posted by dtae on 25 January 2011 - 08:36 AM in PCR, RT-PCR and Real-Time PCR

A few ideas:
- be careful of well position effects - I think I covered this in other areas of this thread or in other submissions to this message board.
- How do your T/S ratios look comparing between multiplex and singleplex? That is more important than Ct values directly
- What are you CVs like across replicates?

D



#98437 qPCR for telomere length measurement - efficiency issues

Posted by dtae on 24 January 2011 - 05:48 PM in PCR, RT-PCR and Real-Time PCR

How do your standard curves and CVs look using both multiplex and singleplex versions? Are your melt curves good? Is there several cycles seperating the Cq values in T and S in multiplex?

Dan


I meant independently of Cawthon, just to make sure. Did you have to supplement your multiplex with anything or did you use the exact same concentrations/conditions for the singleplex as the multiplex and get a good correlation? I'm not sure why I'm not getting a great correlation so I wanted to hear if others did anything differently.

Thanks!




#98425 qPCR for telomere length measurement - efficiency issues

Posted by dtae on 24 January 2011 - 02:37 PM in PCR, RT-PCR and Real-Time PCR

I believe Cawthon did in his paper..and I've found a nice strong correlation as well.


Has anyone validated the Multiplex by comparing Ct values to the Singleplex Ct values for each primer pair?




#97861 betaine storage - how thermo-stable is it?

Posted by dtae on 18 January 2011 - 10:51 AM in Molecular Biology

Hello,

I recently received some 5M betaine solution (in H20) with the dry ice already gone due to an addressing error on the companies part. I am working to get the company to send out a complementary replacement, but in the meantime am curious whether the Betaine is ok or not? How temperature sensitive is betaine solution? It arrived cold, but with no dry ice left. According to USB, who sent the solution, it is supposed to be stored at -20. However, Sigma's webpage states that it should be stored at +2-8 (http://www.sigmaaldr...=0&QS=ON&F=SPEC) and Q-Bio-Gene (http://www.qbiogene..../pcr/dmso.shtml) says it should be stored at room temp. I plan to use it for a qPCR assay.

Anyone have any experience with the thermostability of betaine solution?

Thanks,
Dan



#97573 qPCR for telomere length measurement - efficiency issues

Posted by dtae on 14 January 2011 - 02:07 PM in PCR, RT-PCR and Real-Time PCR

I've had best luck with the albumin primers from the originally published paper. I forget right now if I tried both of the others, or just the newest one. Avoid the beta-globin primers except for cross-validation (they have a mess of SNPs in them and I think are found in multiple copies across the genome).

d


The products are distinct, however the telomere amplicon melting peak is lower than it should be for samples. In a previous post where Cawthon's direct response was quoted, he explained how this occurs due to the GC-rich scg product stealing the SYBR Green.

Just to clarify, which "old" primers worked the best for you? The ones published in his paper or the first "new" set of albumin primers previously posted in this forum (albugcr1, albdgcr1)? The "newest" primers I'm referring to with this problem were labeled albugcr2, albdgcr2.


I forget what the problem was, but I got better results with the old albumin primers than the new ones. Are the two products distinct on the melt-curve?


I have tried Cawthon's multiplex qpcr method with his newest albumin primers and thermal cycling profile and have found that the albumin gene amplification overlaps with the telomere amplification instead of rising above threshold at a later cycle number than the telomere. In his 2009 paper, the purpose of the multiplex was to thermally separate the two amplifications in the same reaction well, but with these newest primers, albumin amplification occurs at the same cycle number as the telomere and crosses the threshold at the same time as well. Has anyone else used the newest albumin primers posted on this forum and had this problem at all?

I appreciate any response/help. Thanks in advance.




#97568 qPCR for telomere length measurement - efficiency issues

Posted by dtae on 14 January 2011 - 01:50 PM in PCR, RT-PCR and Real-Time PCR

I forget what the problem was, but I got better results with the old albumin primers than the new ones. Are the two products distinct on the melt-curve?

I have tried Cawthon's multiplex qpcr method with his newest albumin primers and thermal cycling profile and have found that the albumin gene amplification overlaps with the telomere amplification instead of rising above threshold at a later cycle number than the telomere. In his 2009 paper, the purpose of the multiplex was to thermally separate the two amplifications in the same reaction well, but with these newest primers, albumin amplification occurs at the same cycle number as the telomere and crosses the threshold at the same time as well. Has anyone else used the newest albumin primers posted on this forum and had this problem at all?

I appreciate any response/help. Thanks in advance.




#96286 Clinical Real-time PCR assay

Posted by dtae on 30 December 2010 - 03:45 PM in PCR, RT-PCR and Real-Time PCR

The slope gives you an estimate of the PCR efficiency. Efficiencies over 100% (more than doubling every cycle) suggests contamination. Less than 100% suggests the reaction is working less well with each cycle. However, efficiencies estimated from the slope of a standard curve are also changed by inhibitors/enhancers in the standard curve dilution.
Efficiencies can also be determined at the individual reaction level using programs such as LinRegPCR.



#96285 NTC appear in real time pcr

Posted by dtae on 30 December 2010 - 03:36 PM in PCR, RT-PCR and Real-Time PCR

also see http://www.protocol-...o-prepare-qpcr/
There someone suggests that it might be contamination of the primers.



#96284 NTC appear in real time pcr

Posted by dtae on 30 December 2010 - 03:27 PM in PCR, RT-PCR and Real-Time PCR

Unless you want to detect levels of gene expression 2^10x (or 1024x) less than when your target normally comes up, sounds like this is not really a problem.

The melting point of a product is determined by its base composition and size. It seems quite possible that primer-dimers could have the same melt point as your product. You could run it out on a gel to see if the size is also the same, or sequence it to see if it is a primer dimer of the target product.

D



#96150 96 well plate plan that converts into a list automatically

Posted by dtae on 29 December 2010 - 02:31 PM in PCR, RT-PCR and Real-Time PCR

Figured it out. Put your sample IDs up top and they will appear in list format below. Its locked without a password to keep us from messing it up.

See attached

Attached Files




#96144 96 well plate plan that converts into a list automatically

Posted by dtae on 29 December 2010 - 02:13 PM in PCR, RT-PCR and Real-Time PCR

I want to be able to enter identification numbers for the contents I have in every well of a 96 well plate on a 8 by 12 grid and then automatically get a two column list of of each well number and its content identification number (e.g. one column with well number: A1, B1, C1, D1...., and another with the respective contents identification number). Anyone know an easy way to do this? Perhaps an excel macro of some sorts?

Thanks,
D



#96141 Translate between 384 and 96 well plate formats

Posted by dtae on 29 December 2010 - 02:08 PM in PCR, RT-PCR and Real-Time PCR

I transfer samples between 96 and 384 well plates using 8 channel pippetman and couldn't find an easy conversion table for what wells the different quadrants of a 384 well plate map to on a 96 well plate. I created an excel file which seems to do the trick for me. I'm posting it here in case it is helpful for others. Simply enter the wells you need converted in Column A (I already have all 384 wells listed, but you can replace them with what you need) and then the 96 well equivalent will appear in the Quad1-Quad4 columns (H-K).
Note that what you enter in Column A is case sensitive.

I am sure there are more elegant ways to do the job and encourage others to post better methods.

Hope this is helpful to someone.

D

Attached Files




#94590 high-throughput retrieval of low volume samples from large well plate

Posted by dtae on 11 December 2010 - 08:34 AM in General Lab Techniques

I have 40 500ul 96-well storage plates filled with 20ul DNA samples in each well. The problem I am having is getting a nice high-throughput method to pull 5ul of all of these samples. After spinning down the plate, the liquid tends to pool at the edges of the wells for most wells, but near the bottom of the well for the central wells (the plate is presumably not flat, or bending when it is being spun down). Using an AP96 (96 channel robot) to try to pull 5ul out of this, we consistently have a few missed wells on each plate. Using a 12-channel pippet yields a similar problem--it is hard to line up all of the tips so they retrieve the volume they are supposed to. I don't want to have to use a single channel to pull all of these samples. Any suggestions?

Thanks,
D



#93972 Using the same PCR plate in more runs

Posted by dtae on 03 December 2010 - 05:57 PM in PCR, RT-PCR and Real-Time PCR

my guess is temperature changes to the plate as well. in particular the light might refract around in some weird way after the deformation. i'd be curious to know if it occurs using white plates (if available for the lightcycler)



#93754 qPCR for telomere length measurement - efficiency issues

Posted by dtae on 01 December 2010 - 09:25 PM in PCR, RT-PCR and Real-Time PCR

see here for more info on what i mean about well-position effects:
http://www.protocol-...ffects-in-qpcr/



#93753 need to know minimum amount of template DNA needed for pcr amplification

Posted by dtae on 01 December 2010 - 09:21 PM in PCR, RT-PCR and Real-Time PCR

not sure--but here are a few hints to start thinking about it:

-a new technique called digital PCR tries to utilize the point which the dilution of template is so low that some number of wells won't amplify. if you look up the literature on that technique i suspect you can calculate the theoretical point at which their is likely to be less than 1 copy your template in the reaction

-if you are trying to get by on really low amounts of template, you might try increasing your number of cycles, or even doing a nested PCR

-you could just test this empirically by creating a standard curve




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