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i dnt think there is a big difference between these two methods provided if you have a proper controls, however, restriction digestion is easier and to some extent you can avoid false positives/negatives compared to PCR. so always my first choice would be enzyme digestion........
good luck..
#153089 Screening for ligation and transformation result
Posted
on 27 March 2013 - 10:31 PM
#152156 Using RT-PCR to find the presence of a deletion in a gene
Posted
on 13 March 2013 - 03:45 AM
since you say deletion prone region is specific and if it is also small and distinguishable in terms of its size by PCR, how about designing primer covering few hundred base pairs at the 5 and 3' end of ur deletion region(which shd present both in wild type and the deletion), so that you can distinguish the presence of deletion by size, in this case even if the deletion is in one allele you shd get two products with size corresponding to deletion and wild type.
#152012 Help for pGEM-T easy vector ligation problem
Posted
on 11 March 2013 - 04:28 AM
make sure your Polymerase have terminal transferase activity.....otherwise you just gotto optimize the vector and insert ratio....sometimes purifying the PCR product helps....
#151773 Western Blot - Protein Marker Present on Film After Developing
Posted
on 07 March 2013 - 12:51 PM
just reduce the quantity of marker you loading, you can reduce its signal, alternatively if you have extra well just skip a well and start loading the samples...may be your target antibody might not be picking signal in a short time, so your longer incubation and more marker loaded are the possible reason for this.. definitely your actin shd be good, so you wnt get signal dominance frm the marker...
#112862 Primer dilution --> problem..
Posted
on 17 June 2011 - 12:09 PM
now its clear to me, you have 27.3 nmol primer right?? so if you dilute your primer with 273 microlitres of water you get 100 Micromolar final conc. this will be your stock (so your dilution is right, may be i got confused with the pmol notation)...now you can make your working concentration 2.5 micromolar.
To get the working conc of 2.5 micromolar you have to take 2.5 microlitres from the stock + 97.5 H2O/T.E whatever you prefer.
And yes if you add 6.4 microlitre from the above working your final primer conc in 20 mcirolitre rxn would be 0.8 micro molar , but are you sure you have to use 0.8 micro molar of each primer (F and R)?. better have a check over it and proceed...
hope this helps.
To get the working conc of 2.5 micromolar you have to take 2.5 microlitres from the stock + 97.5 H2O/T.E whatever you prefer.
And yes if you add 6.4 microlitre from the above working your final primer conc in 20 mcirolitre rxn would be 0.8 micro molar , but are you sure you have to use 0.8 micro molar of each primer (F and R)?. better have a check over it and proceed...
hope this helps.
#112330 Please help! no PCR bands+Description of my PCR protocol provided :(
Posted
on 11 June 2011 - 04:17 AM
have you done control PCR (actin or some known working primers) in the same cDNA?, doing so you can rule out there is no prob with the cDNA, then never stick to the same annealing temperature, the melting temperature of your primer will differ in the reaction mix so it is advisable to do a gradient pcr once you didnt get the amplication in a single reaction. i would suggest you to increase your annealing temperature further more (minimum 65) for your range of tm. Finally check the GC content of your region of interest if it seems to be more than 70 percent you may have to add some adjuvants like DMSO.
Gnana...
Gnana...
