Springer Lab: TransformationYeast96well

From OpenWetWare

Jump to: navigation, search

Back to Springer Lab

1. Grow overnight in deep (1.5mL) 96 well plate

2. Dilute between 1:20 and 1:50 (in practice 1:40) and grow for 4 hours

3. Harvest cultures by centrifugation at 3000 rpm for 5 min

3a. combine pellets from multiple plates to increase cell numbers

4. Resuspend in 1mL of sterile water

5. Wash pellet three times in water (spin at 3 000 rpm between washes).

6. Wash pellets three times in 0.1M LiAc

6a. After last wash, keep 35 microL 0.1 M LiAc in each well

7. Prepare transformation mix per well (see table below)

 240 uL PEG (50% w/v)
 35 uL LiAc (1 M)
 10 uL Salmon Sperm Carrier DNA (5mg/mL)
 1 ug transforming DNA (in 30-50 microL)

8.Add transformation mix to each well with multichannel pipette

9. Vortex cells for 1 min

10. Incubate cells at 30C for 30 minutes

11. Heat shock cells at 42C for 25 minutes.

12. Spin cells down at 3 000 rpm for 5 minutes. Remove supernatant with multichannel pipette.

13. Add 600 microL of YPD per well and incubate overnight at 30C

14. Plate cell suspension on appropriate selection media and isolate transformants after 2-3 days

' 1 well 12 24 36 48 60 72 80 84 96
PEG 50%240288057608640115201440017280192002016023040
LiAc (1M)354208401260168021002520280029403360
Salmon Sperm DNA (5 mg/mL)10120240360480600720800840960
transforming DNA10120240360480600720800840960
Total in microL2953540708010620141601770021240236002478028320
Total in mL0.2953.547.0810.6214.1617.721.2423.624.7828.32


For the actual master mix use 10% more reagents 1 well 12 24 36 48 60 72 84 84 96
PEG 50%264316863369504126721584019008211202217625344
LiAc (1M)38.54629241386184823102772308032343696
Salmon Sperm DNA (5 mg/mL)111322643965286607928809241056
transforming DNA111322643965286607928809241056
Total in microL324.53894778811682155761947023364259602725831152
Total in mL0.32453.8947.78811.68215.57619.4723.36425.9627.25831.152
Personal tools