what happened to the blood samples - (Sep/08/2008 )

in our lab i found a human blood samples stored at 4 C for more than 4 months .
the blood become very dark and contain red clots

what happened to the blood???
and does that affect yield of DNA extracted???

best regards

-Jehane-

The blood is hemolyzed (the cell breakdown) so the enzymes were free into solution the dna yield will be lower than the fresh or frozen blood and if using a spin column will see the eluent with a strange color.

-merlav-

I guess U should not try to find a pellet in this protocol as the cells would have lysed.

-Bungalow Boy-

QUOTE (Bungalow Boy @ Sep 8 2008, 07:24 AM)

I guess U should not try to find a pellet in this protocol as the cells would have lysed.

my metod like this in the link but during washing step the pellet is too large and brownish in colour ever after several washes.

but i wonder the the concentration of dna at 260 is very high and seen clearly on agarose gel but it doesn't dissolve completely in TE buffer and after doing |PCR the specific band appear but very very thin

PLZ help to solve my problem

best regards

-Jehane-

if you phenol chloroform the DNA solution, you should be able to clean it up better. Although there are limits, and sometimes you will have to live with an off colour DNA sample.

As for the PCR, I think there are contaminating compounds in the DNA sample inhibiting the PCR reaction. Cleaning up the DNA sample should improve the PCR reaction. You could also use a diluted DNA sample (1:20 to 1:50) as a template. And that should improve the yields. (PCR is a very fantastic amplification reaction)

-perneseblue-

QUOTE (perneseblue @ Sep 8 2008, 07:33 AM)

Cleaning up the DNA sample should improve the PCR reaction. You could also use a diluted DNA sample (1:20 to 1:50) as a template.

Thank u so much for ur reply but plz explain how i do this dilution and how many ul used in pcr reaction. best regards

-Jehane-

just dilute an aliquot of the DNA solution with TE or sd water.

How many ul of DNA template are you using? I don't have an estimate of the amount of DNA present in the undiluted sample. So how about using a similar volume of diluted template DNA.

-perneseblue-

QUOTE (perneseblue @ Sep 8 2008, 09:06 AM)

i dilute DNA as each 1 ul contain 50 ng DNA
i tried to get 1ul but the bands were so faint
another time i tried to add 2 ul of DNA (100 ul concentration) the bands slightly appeared but so thin.

Best regards

-Jehane-

is this a diagnostic PCR? If so try increasing the number of cycles. Could try increasing the volume of template used.

-perneseblue-

QUOTE (perneseblue @ Sep 8 2008, 03:17 PM)

is this a diagnostic PCR? If so try increasing the number of cycles. Could try increasing the volume of template used.

my cycles are 35-------could i increase more than these?

-Jehane-