GTG Banding

[megha sharma]

1> What is the chemical composition of giemsa stain, either in %,ratio or grams?

2> What is the minimum and maximum humidity required at which slides
can be banded?

3> What is the role of EDTA in Trypsin treatment ( GTG Banding) & what
ratio of EDTA & trypsin used?

4> Why plasma is used in banding when slides takes few seconds of
time, what role it plays. Is there any substitute of it?

5> How can we say that during GTG banding there is no loss of DNA & protein?

6> If humidity has increased from 60 to 70 then at established time &
temp. for blood, abortus(long term) and amniotic slides at 60
humidity, how much time can be exactly increased to get proper bands
at 70 humidity?

7> Can one day old working giemsa stain (by adding to it some amount
of stock stain) can be used on next day if on the previous day it is
used during morning and evening and rest of the time chilled at 4
degree centrigrade?

8> What are the probable factors required to get least precipitation
while staing slides with working giemsa stain?

9> Why methanol is used during giemsa stock stain preparation. How it
helps in active staining of slides?

10> How much day old slides can be easily processed and stained by GTG
banding if it is daily aged and kept covered?

Waiting for reply,
Thank you.

[marg]

Hello megha heaps of good questions!!! ... i have tried answering each
of them:

1> What is the chemical composition of giemsa stain, either in %,ratio
or grams?

we use a 10% giemsa working solution on our slides. To make a 10%
giemsa working solution add 10ml giemsa stock stain ùpto 100ml
Sorensens buffer. Anything between 5-10% is fine also.

2> What is the minimum and maximum humidity required at which slides
can be banded?

I feel temperature is more important we band our slides at room temp,
with a humidity range 45%-65%...it seems to go well.

3> What is the role of EDTA in Trypsin treatment ( GTG Banding) &
what
ratio of EDTA & trypsin used?

not sure about this one????

4> Why plasma is used in banding when slides takes few seconds of
time, what role it plays. Is there any substitute of it?

I think plasma stops the banding action of trypsin by inactivating
the trypsin, you may use 1N NaCl as a rinse instead.

5> How can we say that during GTG banding there is no loss of DNA &
protein?

To my knowlwdge there is no reports in the literature to suppose this
(i maybe wrong though?)

6> If humidity has increased from 60 to 70 then at established time &
temp. for blood, abortus(long term) and amniotic slides at 60
humidity, how much time can be exactly increased to get proper bands
at 70 humidity?

You may discover that banding is not good at these high humidities
because the slide are very moist

7> Can one day old working giemsa stain (by adding to it some amount
of stock stain) can be used on next day if on the previous day it is
used during morning and evening and rest of the time chilled at 4
degree centrigrade?

Working giemsa stain oxidises very quickly in open air, always make a
fresh amount before banding.

8> What are the probable factors required to get least precipitation
while staing slides with working giemsa stain?

Ensure the stain is sourced from good vendors, remove the "green-
blue"film that forms at the top of thestain by blotting it off before
use...if you make your own giemsa ensure the stain is well mixed &
filtered using paper filters.

9> Why methanol is used during giemsa stock stain preparation. How it
helps in active staining of slides?

Methanol from memory is an organic solvent that dissolves the giemsa
powder-glycerol mixture completely over a period of time.

10> How much day old slides can be easily processed and stained by
GTG
banding if it is daily aged and kept covered?

Age stides in a incubator set at 37C and containing a tray of
dessicant for 5-7 days, if problems are encountered with wet slides
try aging slides at 65C overnight in an oven.
We stain 6 to 7 slides in a coplin jar and give us good crisp bands.

[zondervan]

Hi your question (no.6) about high humidity and banding our lab during
humid weather places the places in a drying oven set at 65 degrees C
for 3 hours and we band straight away after remopving the slides from
the oven. The banding is fine 4 us.

On Jun 3, 1:53 pm, megha sharma <ms.ihg....@gmail.com> wrote:

[Lezlie Densmore]

OK, I'll add my 2 cents.

> 3> What is the role of EDTA in Trypsin treatment ( GTG Banding) & what
> ratio of EDTA & trypsin used?

EDTA binds to certain molecules. In this case I believe it's Mg that
it's binding up. Mg inhibits the action of enzymes = trypsin in this
case.

> 4> Why plasma is used in banding when slides takes few seconds of
> time, what role it plays. Is there any substitute of it?

FBS contains alpha-anti-trypsin which inhibits the action of trypsin.
It's also used to stop the action of trypsin when trypsinizing cells
out of flasks.

Lezlie