# 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 😃