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#142418 nonspecific band at 50bp in PCR
Posted
on 28 September 2012 - 05:41 AM
kyakhoob, on 28 September 2012 - 04:03 AM, said:
habglobin
thank you for your suggestion actually i have tried everything. i have added every reagent properly..i tested the DNA and found OK..primers are few month old. my guide told me that the primers might get contaminated. is there any way to know whether the primers are okay or not. please help.
thank you for your suggestion actually i have tried everything. i have added every reagent properly..i tested the DNA and found OK..primers are few month old. my guide told me that the primers might get contaminated. is there any way to know whether the primers are okay or not. please help.
also checking the thermal cycler is an idea, i.e. asking if colleagues have no problems with their PCRs and if your program is still untouched...
#142343 nonspecific band at 50bp in PCR
Posted
on 27 September 2012 - 06:54 AM
Sounds as if this are primer-dimers that occur at this size....i.e. your PCR did not work...perhaps one of the reagents is kaput now (often dNTPs), or DNA degraded, or you forgot to add a reagent to the reaction (Taq, dNTPs, ...)? You have to try out and use a positive control, then you know that the reagents are okay.
#141406 Font for Power Point presentation
Posted
on 13 September 2012 - 12:28 PM
and for the announcement of the results of the replication of the experiment to prove its repeatability, they'll use baskerville? Perhaps then I'll believe it 
#140722 mitochondrial DNA
Posted
on 05 September 2012 - 07:57 AM
I'd say most kits are reliable (I only used a few and did not find much differences, except the price). Salting out works too for mtDNA but the DNA is more "dirty", which is for PCRs usually no problem.
#140425 Is protein degrade by UV ?
Posted
on 31 August 2012 - 09:48 AM
As I wrote it depends on the protein type and intensity (W/m2). In such measurements you don't use the power of a fluorescent lamp...
Anyway for disinfecting it's surely also DNA damage, but as it seems to work also in organisms that shield their DNA with pigmentations or form spores or cysts, so my idea was protein damage (perhaps indirectly via oxidative stress?)
Anyway for disinfecting it's surely also DNA damage, but as it seems to work also in organisms that shield their DNA with pigmentations or form spores or cysts, so my idea was protein damage (perhaps indirectly via oxidative stress?)
#140416 What gel percent and Voltage is needed to separate 2680bp and 2600bp DNA nicely
Posted
on 31 August 2012 - 09:08 AM
I'd agree with gfischer. But if you really have to do it:
High Resolution Agarose, 0,8 - 1%
Lithium borate or Lithium acetate buffer
running distance as long as possible
not long running times (buffers allow higher voltages), e.g. 20-30V/cm
In BioTechniques (2004) 37:598-602 (Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media) you find some more informations how to do it, though you might need to do some adaptation.
High Resolution Agarose, 0,8 - 1%
Lithium borate or Lithium acetate buffer
running distance as long as possible
not long running times (buffers allow higher voltages), e.g. 20-30V/cm
In BioTechniques (2004) 37:598-602 (Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media) you find some more informations how to do it, though you might need to do some adaptation.
#138481 Do you know about reverse pipetting? A survey
Posted
on 29 July 2012 - 03:06 AM
I'd avoid using this technique, because the usual way of pipetting is so internalised and done automatically that then I'd really have to focus what I do to avoid that the normal pipetting mode comes back. Then I'd be confused and a mixed and therefore incorrect mode would happen soon.
So only if I've to pipet glycerol what happens rarely.
So only if I've to pipet glycerol what happens rarely.
#138212 Change in the pH of stock solution
Posted
on 24 July 2012 - 09:16 AM
Or just measuring inaccuracies? As in several threads here mentioned measuring the pH of water is quite tricky and small impurities (e.g. from the storage solution), differences in dissolved CO2, or calibration changes of the pH-meter lead to larger pH changes of the water or different measurements.
Not sure about the buffering capacities of seawater. Is there any?
Not sure about the buffering capacities of seawater. Is there any?
#137085 Phd topic
Posted
on 06 July 2012 - 11:29 AM
desertrose, on 06 July 2012 - 01:10 AM, said:
hobglobin, on 05 July 2012 - 07:48 AM, said:
some more details would be nice...it's hopefully more than just designing a primer pair for the bacterium...
#136976 Phd topic
Posted
on 05 July 2012 - 07:48 AM
some more details would be nice...it's hopefully more than just designing a primer pair for the bacterium...
#136613 DNA extraction from Potato Chips
Posted
on 29 June 2012 - 07:54 AM
did you try out already any protocol? If not I'd just start first with a protocol established in your lab...
My first idea would be to remove as much as oil as possible before e.g. by crushing them to smaller pieces and then putting it on absorptive paper (e.g. kitchen towel)....
My first idea would be to remove as much as oil as possible before e.g. by crushing them to smaller pieces and then putting it on absorptive paper (e.g. kitchen towel)....
#134180 Glucose Assay
Posted
on 08 May 2012 - 06:39 AM
...but calibrated for blood glucose. So if you use them you should test if they are similar sensitive and exact for your samples (there were some substances that I not exactly remember, anyway also other sugars that are perhaps in the sample but never in blood, and then result in false measurements).
#131893 PCR malaria diagnosis nested PCR smear and non specific bands
Posted
on 28 March 2012 - 10:35 AM
Many possibilities and questions...some thoughts:
Why a nested PCR?
Did you check the results of the first PCR alone?
For 205 bp 2min extension time is much too long...15-30 s should be enough (or even less)
Reducing Mg-concentration and cycling numbers might improve specificity
less Taq (0.5 -1 U is also enough usually, reduces possible unwanted amplifications and saves money too)
Try gradient PCR to optimise annealing temperature., i.e. a higher Ta might work also
Not sure if less cycles in the first part of the PCR might help, too
Why a nested PCR?
Did you check the results of the first PCR alone?
For 205 bp 2min extension time is much too long...15-30 s should be enough (or even less)
Reducing Mg-concentration and cycling numbers might improve specificity
less Taq (0.5 -1 U is also enough usually, reduces possible unwanted amplifications and saves money too)
Try gradient PCR to optimise annealing temperature., i.e. a higher Ta might work also
Not sure if less cycles in the first part of the PCR might help, too
#131540 "What People Think I Do / What I Really Do"
Posted
on 22 March 2012 - 01:24 PM
and many funny others...just google them...
