Problems with GTG banding.

[elsalio]

Hello,
i have a few questions about my GTG banding protocol for bone marrows,
because there are times that i cannot see bands.
I age the slides either in 90o C for 1.5h or at room temperature for 3
or more days and then i do the banding.
Firstly, i immerse the slides into trypsin 0.25%-EDTA (1X)
(Invitrogen) for 5-10sec. Should i use trypsin at room temperature or
at 17-18o C?
Then, i immerse the slides into 0.9% NaCl solution and after that in
my Giemsa solution for 5-8 minutes.
I prepare 10% giemsa solution in sorensen's buffer pH 6.8. Where do i
have to store my stock solutions for sorensen, at 4o C or at room
temperature and for how long can i keep the stock solutions?

Any help would be appreciated!!!

[Czepulkowski Barbara (KING'S COLLEGE HOSPITAL NHS FOUNDATION TRUST)]

Dear Elsalio,
Using warm trypsin helps I place the trypsin jar in the incubator for an hour before use then about 10-15 seconds in trypsin depending on the slide. You can test one by putting a coverslip over the finished slide before it dries, then if there are no band you can easily remove the coverslip and de-stain then try again with a longer trypsin time.
We rinse after Trypsin in saline then 6.8ph buffer. Also your stain time seems excessive we stain for no more than 1 minute.

We age slides on a hot plate at 70 degrees for an hour. Nothing ever gets left for days as bone marrow samples are always urgent.

I hope this helps but do use the check for bands with a coverslip mounted in water first then re-staining is much easier!
Barbara Czepulkowski
Consultant Cytogeneticist

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[Majd]

Hello,
Try baking slides at 90c for 2 hours then lower temp to 60c over night.
Trypsin should be prepared fresh daily and be used at room temp. Also make sure the pH of it is within 6.8-7.0 and increase time to 15secs ? Are the chromosome pale? Or solid stained? If pale lower the concentration of trypsin to (.1%)
I would use FBS&Isotonic after trypsin (5ml/45ml)
Then giemsa for 10mins
Good luck

[elsalio]

What about the stock solutions of the buffer, can i keep them at room
temperature or at 4oC? My chromosomes are pale, not solid stained. I
don't know if there is a problem with my giemsa solution. I was using
giemsa (merck) and now i am using giemsa (scharlau). After this change
actually the problem came up.
Thank you very much for your help!!!

[Jernej Kovač]

Hi, 

if you can get merck giemsa try it again, and if there is a change for the better, then you should discharge scharlau giemsa as it is obvous not of good enough quality. Fot he buffers - we store buffer for giemsa at room temperature and giemsa stock also. 

good luck,

J

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[Majd]

Yes you can.
We use Gurr Buffer

Sent from my iPhone

[Czepulkowski Barbara (KING'S COLLEGE HOSPITAL NHS FOUNDATION TRUST)]

Try storing stock solutions at room temperature also try Leishmanns stain instead!


Sent from my iPhone

On 18 May 2012, at 05:30, "elsalio" <ekou...@hotmail.com> wrote:

> What about the stock solutions of the buffer, can i keep them at room
> temperature or at 4oC? My chromosomes are pale, not solid stained. I
> don't know if there is a problem with my giemsa solution. I was using
> giemsa (merck) and now i am using giemsa (scharlau). After this change
> actually the problem came up.
> Thank you very much for your help!!!
>
> On May 16, 10:03 pm, elsalio <ekouv...@hotmail.com> wrote:

> --
> You received this message because you are subscribed to the Google Groups "Cytogenetics methods and trouble-shooting Forum" group.
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> To unsubscribe from this group, send email to cytogenetics-methods-and-t...@googlegroups.com.
> For more options, visit this group at http://groups.google.com/group/cytogenetics-methods-and-trouble-shooting?hl=en-GB.
>

[chromosome artist]

Hi from Canada,

I had a few thoughts about your banding problems.

You can use Trypsin at any temperature, but because its action is
temperature dependent, the warmer it is, the faster it works. If you
heat it to 37 degrees, it will be fatser, but it will slow down as it
cools and your optimal time will change and it will be hard to get
consistent banding. We find leaving it at room temp makes the results
more reproducible.

Even allowing for all that, trypsin time can be widely variable. It
depends on how 'dry' your slides are. It's not scientifically
accurate, but you can think of the aging time allowing the protein
scaffold of the chromatin to collapse and make it more accessible to
the trypsin. Ours can be anywhere from 15 sec to 2 minutes depending
on how they have been treated. If you don't see bands at your usual
time, try doubling it or even tripling it and keep going up until you
do see bands.

I would also decrease your stain time. If your stain is made fresh,
start with 30 sec and also increase that as needed. We use a mixtue
of Leishman's and Giemsa stains and re-make our stain every hour or so
as it fades. I think Giemsa alone doesn't fade this fast, but we like
the bands better with the mix.

You have to really be open to changing your trypsin and stain times
to accommodate the changes in your preps - how they are made (ie light
or dark chromosomes) weather, temp and humidity all affect how your
slides age.

Good Luck!

Melanie

[Sean Zhang]

Z