# 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 bem2d+pBK549 transformants

# 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 😃