We are trying to identify X and Y chromosome in tissue and probe the presence of X chromosome in human tissue by using PCR for specific DNA (for example, ATL1 for X chromosome; SRY for Y chromosome). However, we cannot detect DNA bands for X chromosome after trying to solve many possible technique problems (gel, probe design, separate loading lane, etc). Besides, we find that SRY size differs between tumor and normal tissue consistently. Are we missing something during the experimental processes? Thank you in advance.
Hello, there are several reasons why you are not detecting the X band
I need a little more information to give you more help but with what you mentioned I suggest you
- Did you check the quality of the DNA you use? It may have degraded
- Did you try with other types of probes?
- Probably the design of the PCR cycles has errors
-Check the TAQ polymerase, maybe it was degraded and that is why it is not amplifying
- Regulate the temperature of the cycles you use in the thermal cycler
- Check the time you give in each cycle for alignment, if it is too short the polymerase may not be able to act
ATL1 is from chromosome 14 I think
Are you amplifying the whole gene or just part of it?
Do you use multiple probes for the same gene at the same time? Like a multiplex PCR?
If you need more help you can keep in touch with me and I will gladly support you in any way I can.
Greetings
Christian Octavio González-Villaseñor Thank you and I will check the points you suggest and go back to you later.
Sincerely,
CM
Very good, I hope your PCR works, I'll be on the lookout in case you have any questions, if you need help designing the primers or with the cycles, I'll gladly support you.
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