I have an inducible expression of the enzyme in a newly derived bacterial strain, and methylation may be affected. I want to test methylation efficiency with a bisulfite kit. The kit is the EZ DNA Methylation kit (Zymo Research, D5001).
Do I need to make a special plasmid with a certain sequence for the bisulfite test, or it works more or less equally for any DNA sequence?
You can order specially methylated DNA controls such as Zymo (catalog # D5013). You should not need this though as the kit should work for any DNA sequence. Are you planning on testing via sequencing or qPCR/ddPCR? If so there are aspects of primer design you may want to research before designing primers. Sequencing would not require that though but could be more costly depending on what you are testing.
Dear Joshua Beytebiere , thank you for your kind reply! I was not precise enough in my question, and my wish to keep it short played a bad joke.
I just want to test the methylation state of the DNA in my strain roughly. My plan is:
1. to make a plasmid with a certain sequence insert
2. clone it into a plasmid,
3. transform into bacteria,
4. stimulate expression of my gene, that affects methylation
5. harvest plasmid,
6. make the test with EZ DNA Methylation kit (Zymo Research, D5001)
7. Send 20 clones for Sanger sequencing of the insert.
So I was looking for a special sequence for the insert, that would be my template to test the methylation with bi-sulfite. from your reply I guess the sequence of the "bait" doesn't matter. Thank you!
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