For me it looks like there is no banding pattern at all! Your chromosomes look overtrypsinized! Are you sure the protocoll sais Trypsin Treatment for 8 minutes??? We do the Trypsin Treatment for only a few seconds, depending on Trypsin concentration!

Well, usually GTG-banding is performed at room temperature and slides should also be at room temperature. all Solutions should be renewed every 4-5 hours. The Age of the slides is not vital but has to be considered. The older the slide the longer the Trypsin exposure time. In General, the exposure time should be determined first using a test slide. Usually, the optimal Trypsin exposure time works for a whole batchof slides afterwards. START with 10 seconds, for example. If the banding is not pronounced enough the exposure time has to be increased, if the chromosomes look fuzzy the time has to be reduced!

Trypsin-Stock Solution (30mg/ml): Dissolve 3g Trypsin 1:250 (Difco) in 100 ml 0.9% NaCl (at 37 degrees C for better solubility). Make 1 ml aliquots and store at -20 C.

Trypsin WORKING Solution: Thaw one aliquot of Trypsin Stock Solution and make up to 50 ml with NaCl in a coplin jar. Adjust pH to about 7.5-7.8 !

Giemsa Stain: 7-10% in Phosphate buffer

For me it looks like there is no banding pattern at all! Your chromosomes look overtrypsinized! Are you sure the protocoll sais Trypsin Treatment for 8 minutes??? We do the Trypsin Treatment for only a few seconds, depending on Trypsin concentration!

Well, usually GTG-banding is performed at room temperature and slides should also be at room temperature. all Solutions should be renewed every 4-5 hours. The Age of the slides is not vital but has to be considered. The older the slide the longer the Trypsin exposure time. In General, the exposure time should be determined first using a test slide. Usually, the optimal Trypsin exposure time works for a whole batchof slides afterwards. START with 10 seconds, for example. If the banding is not pronounced enough the exposure time has to be increased, if the chromosomes look fuzzy the time has to be reduced!

Trypsin-Stock Solution (30mg/ml): Dissolve 3g Trypsin 1:250 (Difco) in 100 ml 0.9% NaCl (at 37 degrees C for better solubility). Make 1 ml aliquots and store at -20 C.

Trypsin WORKING Solution: Thaw one aliquot of Trypsin Stock Solution and make up to 50 ml with NaCl in a coplin jar. Adjust pH to about 7.5-7.8 !

Giemsa Stain: 7-10% in Phosphate buffer

I agree with Christian, it looks overtrypsinized. I usually trypsinize the glass max for 30 s. The time depend from the age of your slides (the time of preparation and time of trypsinizing). Ithe older slides, the longer time is needed.. Max I use 1 min for older ones. But also I use cold trypsine solution to make bands.

We use 0,25% Trynsin, cold or room temperature. Time is 15-30 SECONDs! We prefer slides 1-2 days older, without any pretreatment before trypsin. After trynsin - just rinse a slide at 2xSSC and then in 2% Giemsa.

The protocol 1 (heat G-banding) looks more complicated then protocol 2 in Rooney, 2001. Would you consider trying protocol 2 to test your trypsin? We routinely use the protocol similar to  protocol 2 to get G-bands of good contrast.

hi diar  we  used G-banding in our lab at 2008 .

I agree with Svetlana V pavlova ,but with heating  before trypsinization

I have worked in clinical chromosome analysis for more than 15 years. We don't use Giemsa stain in clinical analysis anymore. Because the Giemsa stain is not better than Wright's stain.I suggest the process is (in clinical analysis used)

Age slide under 95℃ for 1 hour. (important)

cooling slide and then trypsinized (0.25g trypsin/100 ml EDTA solution (for 10-40 sec., depends on your room temp)

rinse slide with 95% EtOH to stop the trypsinzation

Using the wright's stain for 30 sec to 1 min---wright's  stain: pH 7.0 buffer (Gurr's)(1 ml: 3 ml) 

rinse with water, dry slide

and your image that I think it's not overtrypsinized. It's undertrypsinzed. This result was found in student's experiment class. Because overtrypsinzed will cause the chromosomes become empty. You can't see the chromosome's structure well. All histones were digested by trypsin, wright's stain or Giemsa stain can't bind to the chromosome by interact with histones. I have answered the question as same as your question, maybe you can find it.

We age the slides 1h at 100 C. Then with trypsinized in a solution of  PO4H2K and NaCL (0.1%trypisn.) The time of trypsinization depends on the room temperature, etc, you need to try till you get the best results of banding. Then wash in NaCl y stain in Wright´s stain 10 minutes.  

We moved from using trypsin to pancreatin which produces nicer bands, you may want to try using pancreatin.

Well, I worked with both during the last 20 years of my chromosome routine work in different labs. Pancreatin is really not that agressive like Trypsin and therefore the bands are somehow "nicer". But using Pancreatin will change the whole protocoll and therefore you start almost at the beginning! One should take this always in mind!

Hi,

With the trypsin method. We usually let the slides age by keeping them in a dry place for at least 7 days after harvest. Then when using the trypsin it is usually used in a watherbath in a coplin jar with just dipping the slides for 40 seconds then put in a buffer for just a rinse.

Your chromosomes looks like the trypsin was not working it is supposed to do so digestions for the chromosomes.