I want to get the lysate of sperm tissue to isolate DNA ,But when I was reading the protocols for tissues lysis, they use DTT in lysis buffer .So I got confused about if the DTT should be added. can I use alternative of the DTT lysis ?
Does someone have the experience about this?
Please tell me the detail about this. Thank you so much.?
Mohammad
Hi Mohammad,
DTT is a reducing agent which is often used to reduce disulfide bonds of proteins. Im guessing this is necessary when working with sperm cells in order to disrupt disulfide bonds of the sperm cell's coating, allowing extraction of the DNA. There are other alternatives, like TCEP (more expensive than DTT) or DTE (more stable than DTT). DTT is however incredibly unstable (half life of ~40 hrs in pH 8.5 soln), so it is best to make the solutions fresh as required.
If you opt not to use the DTT, you might need to resort to physical disruption (sonication) and lysis in order to obtain a high enough yield for downstream applications, but this is also likely to shear the DNA into smaller fragments, so it may not be a viable alternative. What is the intended downstream application?
Kind regards,
Riegardt Johnson
Hi Mohammad,
DTT is a reducing agent which is often used to reduce disulfide bonds of proteins. Im guessing this is necessary when working with sperm cells in order to disrupt disulfide bonds of the sperm cell's coating, allowing extraction of the DNA. There are other alternatives, like TCEP (more expensive than DTT) or DTE (more stable than DTT). DTT is however incredibly unstable (half life of ~40 hrs in pH 8.5 soln), so it is best to make the solutions fresh as required.
If you opt not to use the DTT, you might need to resort to physical disruption (sonication) and lysis in order to obtain a high enough yield for downstream applications, but this is also likely to shear the DNA into smaller fragments, so it may not be a viable alternative. What is the intended downstream application?
Kind regards,
Riegardt Johnson
Hi Riegardt Johnson thank you for your interesting and answer .
actually I want to use RFLP PCR to determine SNPs type In several genes
So I cant find DTT in my lap now and I cant waiting more time to buy it from out side of my country Iraq.
All over , back thank you very much Riegardt Johnson ,
Regardsm
Mohammad Alzeyadi
Hi Mohammed,
If you have access to a sonicator, that should work just fine. Or an alkaline lysis buffer with simple vortexing could even work. Since your amplifying DNA through PCR, you should be ok with slightly sheared DNA. The yield might be slightly lower, but again, the PCR should take care of that!
Good luck!
Hi Mr Riegardt Johnson. may I ask, which alkaline lysis buffer that can be used for extraction of sperm DNA?
Riegardt Johnson
- Badges
- Science method