23. Title : Genomic extractions,PCR for checking and sequencing of dbem3 and dbem3dnrp1 new strains#

23.1. Date#

12022021-19022021

23.2. Objective#

  • To check through PCR using gDNA as template if those strains are correct.

23.3. Method#

  • Use the same protocol and experience gather from when I extracted gDNA from ywt04

  • 12022021:

    • Inoculation on YPD+6x+NAT

  • 15022021:

    • 5 eppies from the tube , pulling 2mL of the culture.

    • 30uL elution buffer per eppi

23.4. Results#

  • PCR at 65C annealing T.

  • 16022021: PCR with primers oLL401/402 and SPY1/2 , together with a positive control. THEY ARE ALL GOOD

  • One comment is that for the biological replicate 2 is very faintly shown a band in the size of bem3 , but most of the DNA have NAT in the right location.

    • Do serial dilution in plate to clean the stock

      • No growth of ylic137(2) after 48h of growth (??)

  • PCR and genomic extraction of ylic138

  • Try a PCR with 60C annealing temperature instead of 65C

    • It worked better the PCR for ylic138 with 60C annealing temperature!

  • Streak glycerol stocks from ylic137_1, 2 , ylci138_1 and 2 in YPD+6x+NAT and YPD+6x+NAT+HYGRO respectively to double check the phenotype of them. 19022021

  • PCR of ylic137_1 and 2 and ylci138_1 and 2 to send to sequence

    • All PCR products are correct yet still ylic137_2 has this band on BEM3 length. However its concentration is really low compared to the other product (100ng/uL vs 4ng/uL, measured in gel)

    • PCR purification: 20uL elution buffer

  • Send samples to sequence (19022021)

23.5. Conclusion#

  • The sequencing for the two biological replicates (I and II) from ylic138 seems to be correct. Also for the case of ylic137I.

  • The sequencing from ylci137_II did not work. For primer oLL402 I got not alignment and for oLL401 is blank. I suspect that could be an error when I was preparing the samples .

  • I will clean the ylic137_II and rerun all the checks again in the new colonies.