how much trypsin should i dilute in PBS for g banding? i also cant understand if i let my slide to much time in trypsin or not. Can anyone show me a picture of overtrypsinised chromosomes and undertrypsinised to see the difference of the image?
Trypsin Stock Solution: Dissolve 1g of Trypsin (250,000 USP units/gm) in 100ml of DPBS (without Ca++ and Mg++). We usally make the stock solution and freeze it in 6.5ml aliquots.
Trypsin Working Solution: add 6.5ml of trypsin stock solution to 250ml of DPBS (without Ca++ and Mg++). The trypsin working solution can stay at room temperature for up to 2 hours.
We dip our slides in the trypsin working solution between 10-15 secsonds, rinse in DPBS and them immerse them in the stain.
We have switched from Trypsin to Pancreatin because we felt we were getting better banding resolution.
Pancreatin Stock Solution: Dissolve 50g of Pancreatin (4 × USP) with 17g of NaCl powder into 2000ml of molecular grade water. Stir up to 1 hour at fast setting. Make 15ml aliquots of the pancreatin stock solution and freeze
Pancreatin Working solution: add 15ml of pancreatin stock solution to 235ml if HBSS (not DPBS) (without Ca++ or Mg++).
We immerse the slides in the pancreatin working solution for 10-15 seconds. Rinse in DPBS and immerse in the stain.
I feel great banding usually results only when you have nicely spread,gray, flat metaphases from the slide making process
I have attached a few pictures from an old, but great book, that is essential for anyone performing cytogenetic analysis.
http://www.sigmaaldrich.com/catalog/product/sigma/p3292?lang=en®ion=US
https://www.fishersci.com/shop/products/trypsin-10/s25839
http://www.fishersafety.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalogId=29104&productId=2987056&distype=0&fromSearch=0&hasPromo=0
Thank you vary much for your help. i will try your protocol.
i use 1 ml 0.25% trypsin/EDTA in 45 ml PBS. Is the trypsin concentration to low??
If your banding is coming out dark (no banding pattern) it is possible the concentration is to low. I beleive using Trypsin with EDTA is an old method, i'm not sure how many other labs are using trypsin/EDTA. I think the pancreatin method has worked extremely well for us.
iS THERE ANY PROBLEM USING PBS INSTEAD HBSS? THE WORKING SOLUTION CONTAINING 5ml of pancreatin stock solution AND 235ml HBSS IS PRESERVED IN THE FRIDGE? HOW MUCH OF THESE SOLUTION DO YOU USE FOR EACH SLIDE?
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