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

# Date

10072020

# Objective

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

# 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

# Sanity check

• [x] 16072020 - Growth in -URA and few colonies in -ADE

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

• [x] 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.

• [x] 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

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

# Results

• I have some colonies to proceed with SATAY 😃