Title: 03102019- III pBK549 transformation on ylic133 for further sanity check in SATAY :blush: :punch:

64. Title: 03102019- III pBK549 transformation on ylic133 for further sanity check in SATAY :blush: :punch:#

64.1. Date#

03102019-09102019

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.

14:00 Incubation form glycerol stocks of ylic133_1, ylic133_4,ylic133_5 and Byk832 in new YPD+6xADE media.
New pBK549 plasmid extraction from bacteria. Miniprep and enzyme restriction assay in the same day of transformation. Look here for the restriction protocol
04102019 9:15 OD measurements

	OD-10X dilution	Titer	Dilution factor to OD=0.5	Time
ylic133_1	0.368	3.68	7.76X	9:15
ylic133_5	0.478	4.78	9.56X	9:15
Byk832	0.057	0.57	1,14X	9:15

Miniprep to extract pBK549
- concentration: 46,7 ng/ul total volume 240 uL
- Digestion testing by EcoRv, PvuII and both:
13:00 OD measurements 10x dilution

	OD-10X dilution	Titer	Ready to transform	Time
ylic133_1	0.255	2.5	Yes	9:15
ylic133_5	0.198	1.98	Yes	9:15
Byk832	0.385	3.8	Yes	9:15

3ul plasmid (46,7ng/ul) implies 140ng plasmid.
I prepare another stock of 1M of LiAc.
Plating 150ul cells+50ul MiliQ and 30ul cells+170ul MiliQ in -URA+6xADE and 20ul cells +180ul MiliQ in YPD (positive control)

09102019 This time the transformation really works , giving plenty of colonies in all the strains. I suspected that what made the big difference is the change of the LiAc.
The negative control is good, so there is no growth in any of the plates.