leelee

if it doesn't react to anti-a, anti-b or anti-ab (therefore, absence of antigen), and the choices are abo, then what does that leave?

You can calculate all day, and look up any number of formulas, but that won't make your PCR work. Try it. I'd suggest starting with a 55 anneal.

And I just see that leelee stated the same thing in a lot less words

Dear Science noob,

This is an extremely common problem that I have seen over the years. You comment:-

"methods provided in published papers"    .............. this is a big problem in that this part of a paper should probably be the largest section but is commonly just cut to a bear minimum. There are lots of "tricks" that are left out of this section that are essential for thew experiment to be reproduced.

"Same concentration, same cell lines, same conditions"................every cell paper I read states " the cells were grown in a humidified CO2 Incubator at 5% CO2. How many researchers routinely check for humidifiaction AND FYRITE THEIR INCUBATORS TO CHECK PRECISELY THE INTERNAL CO2 CONCENTRATION IN THEIR INCUBATORS.

ARE THE INCUBATORS DIRECT HEAT OR FAN ASSISTED?

WHERE DOE YOUR FOETAL CALF SERUM COME FROM?.......there are massive differences between the qualitof serum sourced for the many countries of the world that produce it.

HOW MANY RESEARCHERS HAVE AUTHENTICATED THEIR CELLS BEFORE USING THEM?

HOW MANY RESEARCHERS ARE WORKING WITH MYCOPLASMA INFECTED CELLS BECAUSE THEY DO NOT TEST THEM?


Does this start to answer your question about reproducibility?

Kindest regards

Uncle Rhombus

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.

You just registered to ask this question? I'm impressed
let's say you want 1ml of 0.4%
c1v1=c2v2
0.6xv1=0.004x1000ul =>v1= 6.6 ul, take this amount from the original bottle and top up to 1 ml with water.

The data sheet you attached says:  "These Primers and Probe are synthesised and purified by PAGE. They are supplied as 0.2 OD (6.6 μgs) lyophilized. To reconstitute, dissolve in 20 μl sterile water, check concentraion by OD before use."

So, according to this you just have to add 20µl of water.  The predicted concentration will be 330ng/µl  (6.6µg/20µl), but you should confirm by nanodrop or similar methods.   From this dilute to your working concentration.  

I'm not sure where you get that  "The lyophilized concentration of primer is 20 pmol"  On that, a very important point.  20pmol  is NOT a concentration, is total amount.  Same goes for 7µg  (or actually 6.6µg according to the attached document), this is NOT a concentration but an absolute amount.   You can make this up to the required concentrations by adding water (or TE).

Back to what the data sheet suggests, of dissolving in 20µl of water, and assuming that 6.6µg of primers are indeed 20pmol.  Primers dissolved this way will be at a concentration of 1µM  (20pmol/20µl).

Hope this helps.

It doesn't run faster if you have too much, if anything it is more likely to run slower (e,g. get caught in the well).

Hmmm, if they were immortalized - how did they do it?   It is quite likely that they used an insert like large T antigen, in which case the MEFs may have lost it (and they certainly won't be passage 2, more like passage 30 since the initial isolation and having to get the gene stably expressed).   You may need to find out how they did the immortalization and either re-do it or try adding some of the selective antibiotic to the medium to ensure that the immortalizing agent's expression is maintained.

@ leelee

SYBR Green  for example..

You should be able to find them yourself....
However keep in mind that some of the alternatives seem to be more "dangerous" then ethidium bromide itself.

PS. the horror stories about ethidiumbromide are really over the top and not true at all. Many people seem not to be able to search for information themself and just accept "stories" others told them.

Another possibility is that you have contaminated the cells during your competent cell prep with something other than E. coli.  Tap water or ice often contains resistant organisms.

First get rid of erythrocytes. There is huge amount of them and heme is a potent PCR inhibitor. We lyse the erythrocytes from whole EDTA blood (heparin is also not suitable for PCR later) and use only washed leukocytes for DNA isolation.

5-9 ml of blood fil up to 50 ml with 1x lysis buffer (ice cold) and mix.
40 minutes on ice, mix gently once in a while.
centrifuge 10 minutes/ 1125 g/ 4 deg
draw off supernatant, wash (resuspend pelet) with 15 ml of 1x lysis buffer
centrifuge again
wash with 15 ml ice cold PBS pH 7.4
centrifuge again
now you should have (mostly) leukocytes, for any subsequent DNA isolation method.

10x lysis buffer
1.5 M  NH₄Cl   
100 mM NH₄HCO₃   
10 mM Na₂EDTA
adjust pH to 8.

But if you use QIAGEN kits you may as well buy QIAGEN kit designed for blood, then you don't need to do this.

science noob, on 16 December 2012 - 10:38 PM, said:

I use this system - any time the cells are lifted and expanded or for freezing down add a passage #.  For instance if I took some cells at p2 and lifted them and froze them down (p+1), I would write p3 on the tube and thaw them as p3...

Also note that the different serum/DMSO/medium combinations should all work fine, though I think most people would agree that 10% DMSO and a slow freeze with a quick thaw for revival is best.  Adding DMSO straight to serum can cause precipitation of proteins in the serum, so I tend to avoid that and use 10% serum (Actually this owrks out ot be about 12% I think, with the serum that is already in the medium), 10% DMSO and 80% medium.

here