65. Title : 01102019- II pBK549 transformation on ylic133 for further sanity check in SATAY :pensive:

65.1. Date

01102019-03102019

65.2. 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.

65.3. Method

• 30092019 -Incubation of single colonies from the plates from 24092019 and Byk832 as positive control from glycerol stock at 10:30.

• 01102019 - OD measurements at 9:35

	OD-10X dilution	Titer	Dilution factor to OD=0.5	Time
ylic133_1	1.214	12.14	24	at 9:35
ylic133_4	1.216	12.16	24	at 9:35
ylic133_5	0.316	3.16	6	at 9:35
Byk832	0.308	3.08	6	at 9:35

• at 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	at 13:15
ylic133_4	0.118	1.18	close to OD=2	at 13:15
ylic133_5	0.233	2.33	yes	at 13:15
Byk832	0.290	2.9	yes	at 13:15

• at 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.

65.4. Results

• 031022019 No clonies in the selection plates :pensive: 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.

65.5. 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.