I am using SSC buffer to make a giemsa (20mg per 50 ml SSC buffer). we use aged slide (65 degree for overnight) for treating with Trypsin Solution for 20 sec at 20 degree. We get minor banding pattern, but not able to get sharp and clear banding.
Slide aging, Trypsin, and Giemsa must all work together. The concentration of your Trypsin is also important. Too strong or too weak with also result in poor banding, and the type of rinsing between the Trypsin exposure and the Giemsa exposure will have some effect also. The Trypsin must be freshly diluted also, or poor banding will be seen. Our protocol is to use Trypsin at 0.0025% ('yes', quite low), heated to 30'C for 3-4 minutes, then rinsed with 70% Ethanol, and then treat with fresh "5% Giemsa" stain [prepare 5% Giemsa stain by diluting 25 mL Giemsa (Millapore-Sigma PN 1092040500) with 500 mL 1X PBS (from ADS Biotec)], Giemsa stain immersion for 5 to 7 minutes. These are much longer times than many typical labs use, but we find they are more flexible and easier to 'tune-in' than most short protocols that use more concentrated Trypsin and Giemsa. It is also designed to work with our special aging device that completes slide aging in 20-30 seconds rather than overnight, but we have found that it works OK for the longer time 'oven-baked' aging also.
Thank you David Hild,
kindly inform me that, trypsin should be made in what...???
We dilute this with 1XPBS as follows. For the concentrated Trypsin (2.5%), we normally aliquot this out upon receipt as 1.5mL into 2mL snap top tubes and freeze until ready for use. Then when time to use, we thaw a tube, take 50mL of 20X PBS concentrate, dilute that with ~900mL DI water, add the trypsin and dilute to 1L for use. We verify pH is 7.35-7.50, and adjust with 1N HCl or 1N NaOH as needed to reach that pH range.
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