Springer Lab: TransformationYeast96well

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(New page: 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. ...)
Line 34: Line 34:
14. Plate cell suspension on appropriate selection media and isolate transformants after 2-3 days
14. Plate cell suspension on appropriate selection media and isolate transformants after 2-3 days
 +
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|'''1 well'''
 +
| align="center" style="background:#f0f0f0;"|'''12'''
 +
| align="center" style="background:#f0f0f0;"|'''24'''
 +
| align="center" style="background:#f0f0f0;"|'''36'''
 +
| align="center" style="background:#f0f0f0;"|'''48'''
 +
| align="center" style="background:#f0f0f0;"|'''60'''
 +
| align="center" style="background:#f0f0f0;"|'''72'''
 +
| align="center" style="background:#f0f0f0;"|'''80'''
 +
| align="center" style="background:#f0f0f0;"|'''84'''
 +
| align="center" style="background:#f0f0f0;"|'''96'''
 +
|-
 +
| PEG 50%||240||2880||5760||8640||11520||14400||17280||19200||20160||23040
 +
|-
 +
| LiAc (1M)||35||420||840||1260||1680||2100||2520||2800||2940||3360
 +
|-
 +
| Salmon Sperm DNA (5 mg/mL)||10||120||240||360||480||600||720||800||840||960
 +
|-
 +
| transforming DNA||10||120||240||360||480||600||720||800||840||960
 +
|-
 +
| Total in microL||295||3540||7080||10620||14160||17700||21240||23600||24780||28320
 +
|-
 +
| Total in mL||0.295||3.54||7.08||10.62||14.16||17.7||21.24||23.6||24.78||28.32
 +
|}
 +
 +
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''For the actual master mix use 10% more reagents'''
 +
| align="center" style="background:#f0f0f0;"|'''1 well'''
 +
| align="center" style="background:#f0f0f0;"|'''12'''
 +
| align="center" style="background:#f0f0f0;"|'''24'''
 +
| align="center" style="background:#f0f0f0;"|'''36'''
 +
| align="center" style="background:#f0f0f0;"|'''48'''
 +
| align="center" style="background:#f0f0f0;"|'''60'''
 +
| align="center" style="background:#f0f0f0;"|'''72'''
 +
| align="center" style="background:#f0f0f0;"|'''84'''
 +
| align="center" style="background:#f0f0f0;"|'''84'''
 +
| align="center" style="background:#f0f0f0;"|'''96'''
 +
|-
 +
| PEG 50%||264||3168||6336||9504||12672||15840||19008||21120||22176||25344
 +
|-
 +
| LiAc (1M)||38.5||462||924||1386||1848||2310||2772||3080||3234||3696
 +
|-
 +
| Salmon Sperm DNA (5 mg/mL)||11||132||264||396||528||660||792||880||924||1056
 +
|-
 +
| transforming DNA||11||132||264||396||528||660||792||880||924||1056
 +
|-
 +
| Total in microL||324.5||3894||7788||11682||15576||19470||23364||25960||27258||31152
 +
|-
 +
| Total in mL||0.3245||3.894||7.788||11.682||15.576||19.47||23.364||25.96||27.258||31.152
 +
|}

Revision as of 17:35, 14 April 2011

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 (10mg/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