# Title : 01102019- II pBK549 transformation on ylic133 for further sanity check in SATAY 😔

# Date

01102019-03102019

# Objective

To ensure that the constructed strain is able to pass the Satay sanity check, and then I can continue with the further steps, like mating with yEK7a.

# Method

  • 30092019 -Incubation of single colonies from the plates from 24092019 and Byk832 as positive control from glycerol stock @10:30.
  • 01102019 - OD measurements @9:35
OD-10X dilution Titer Dilution factor to OD=0.5 Time
ylic133_1 1.214 12.14 24 @9:35
ylic133_4 1.216 12.16 24 @9:35
ylic133_5 0.316 3.16 6 @9:35
Byk832 0.308 3.08 6 @9:35
  • @9:55 Incubation at OD~= 0.5 to OD~=2 in YP+6x ADE
OD-10X dilution Titer Ready to transform Time
ylic133_1 0.121 1.21 close to OD=2 @13:15
ylic133_4 0.118 1.18 close to OD=2 @13:15
ylic133_5 0.233 2.33 yes @13:15
Byk832 0.290 2.9 yes @13:15
  • @14:30 30C incubation
  • 100ng of pBk549
  • Notes on the procedure :
    • I did not vortex the cells with the transformation mix, instead I pippeted in and out after each ingredients, by this way I avoided clumps formation that hinder transformation efficiency.
    • I did not use the maximum speed (13300) of the centrifuge to get rid of the LiAc, instead I used 8000rpm.
    • I heated the ssDNA to 95C and then transfered to ice before I added the 0.1M LiAc , so I could do all steps of LiAc washing and samples separation consecutively.
  • I plated all (200ul) in the selection plates (-URA+6x ADE), both, negative control and transformed cells.

# Results

  • 031022019 No clonies in the selection plates 😔 I just see 3 colonies in ylic133_1 plate.
    • Possible causes:

      • The plasmid is degraded
        • check: I loaded 10ul of plasmid into a gel to see If I have low bands, that could indicate degradation of the plasmid.
        • This result implies that plasmid degradation IS NOT THE CAUSE.
      • The plasmid was lost
        • check: I restreak one colony I had on the selection plate into a new -URA+6xade plate to see if they actually acquired the plasmid:
          • This implies that is neither the cause.
      • I used an extremely low amount of plasmid
        • This could be due that I did not vortex the plasmid before using it, and maybe some of it sediment to the bottom, and effectively I took less plasmid than what I thought. Next time I should vortex it anyways.
      • The selection plates are not good i.e. nothing can grow there.
        • check: I plated a positive control (yWKD017 and ylic132) on the -ura plate+6x Ade I used for the selection plate.
        • This growth of positive control on the plate indicates that the plates ARE NOT THE CAUSE
      • My reagents of the transformation protocol are not good.
        • Maybe my PEG50% is not well... If the next transformation does not work , I will replace all my reagents for new fresh ones.
        • I actually replace the LiAc , I did a new 1M stock of 50mL.

# Conclusion

  • Still I dont know the cause of why my transformation arent working, though I discarded multiple causes.
  • I will try again with new plasmid from another bacteria incubation, and after checking that my plates are indeed correct, to discard those factors.