I co-transformed yeast for Y2H and wanted to confirm the presence of my Gene by PCR. I used alkaline and zymolase lysis.
DNA directly from cell lysis may not works well due to any lysis remain.
If you were not using an extracting kit, you can try serial diluting your template to 10 and 100 times to use.
Even if the DNA was isolated by kits, it would still be good to dilute them with TE or water, since there could still be inhibitor existing.
Its not working for yeast cells . And as my antibodies are not standardized i am unable to finalize western though i can see a band at my expected size
How are you prepping the DNA after cell lysis? I've only tried this a few time but I found a protocol for picking yeast colonies into NaOH (possibly 100 mM, but I forget) and giving 30 sec in the microwave. Spin, neutralise (or at least chuck in some TRIS) and use as template for PCR.
- Badges
- Science method