38. Title : Transformation of ylic135 and ylic136 with pbK549 for further SATAY experiment

38.1. Date

10072020

38.2. Objective

To transform two biological replicates from ylic135 and ylic136 with the plasmid that enable the transposition in SATAY, e.g pbK549

38.3. Method

• Yeast transformation Protocol

• 10072020

  • Inoculation of ylic135_1 (colony 13) and ylic135_2 (colony 16) in 10ml of SCM-leu2+6xade media

  • Inoculation of ylic136_4 (colony 5) and ylic136_5 (colony 7) in 10ml of YPD+HYGRO+6xade media

• 13072020

  • OD measurement and dilution of cell cultures.

	OD (100X dilution)	Real OD	Dilution factor
ylic135_1	0.1180	11.8	~20x
ylic135_2	0.149	14.9	~20X
ylic136_1	0.257	25.7	~40X
ylic136_2	0.264	26.4	~40X

- Incubation at 9:30am. 

Use same conditions as the previous successful transformation

• Check OD at 12:30 (Not ready yet)

	OD (10X dilution)	Real OD
ylic135_1	0.058	0.58
ylic135_2	0.051	0.51
ylic136_1	0.041	0.4
ylic136_2	0.065	0.6

• Check OD at 15:00 (Not ready yet :()

	OD (10X dilution)	Real OD
ylic135_1	0.074	0.74
ylic135_2	0.084	0.84
ylic136_1	0.094	0.94
ylic136_2	0.093	0.93

• Check OD at 16:30 (Ready)

	OD (10X dilution)	Real OD
ylic135_1	0.128	1.3
ylic135_2	0.123	1.2
ylic136_1	0.184	1.8
ylic136_2	0.232	2.32

• 3ul plasmid (46,7ng/ul) implies 140ng plasmid.

• No use of negative controls.

• Plating 150ul cells+50ul MiliQ and 30ul cells+170ul MiliQ in -URA+6xADE and 20ul cells +180ul MiliQ in YPD (positive control)

• 15072020

• Colonies in the -ura+6x ade plate from ylic136: \(\Delta\)nrp1::HYGRO

• Not yet visible colonies from ylic135, \(\Delta\)bem2::leu2

• 16072020

38.3.1. Sanity check

• 16072020 - Growth in -URA and few colonies in -ADE

  • 22072020 : Only one colony from ylic136_1 passed the check, colony 2.

• 22072020 - Plated 8 colonies more per biological replicate to keep screening.

  • 2 more colonies (#14 and # 16) from ylic136_1 passed the sanity check. None from ylic135_1.

• 23072020- Plated 16 more from ylic135_1 and ylic135_2

  • Colony # 11 from ylic135_1 passed the test

  • Colony # 18 from ylic135_2 passed the test

• 07082020- Plate more from ylci35_1 and 2 to at least have 3 technical replicates in one of them for SATAY.

38.4. Results

• I have some colonies to proceed with SATAY :)