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Back to [[Springer Lab]] | Last updated by [[User:Jue Wang|Jue Wang]] 10:53, 19 July 2011 (EDT)
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=YEAST TRANSFORMATION PROTOCOL=


'''YEAST TRANSFORMATION PROTOCOL'''
'''Note:''' This is a simplified (i.e. sloppier) version of a protocol by Gietz. If you need to troubleshoot, refer to the more detailed [http://home.cc.umanitoba.ca/~gietz/method.html original version].


Last updated 20110427
This procedure takes 4-6 hours from start to finish, plus an inoculation the day before.


'''Day 1'''
==Reagents==
Besides culture media, the transformation requires the following reagents:
* Icebox
* Boiled salmon sperm DNA (ssDNA), 10 mg/mL
* Polyethylene glycol (PEG) 3500, 50% w/v
* Lithium acetate (LiAc), 1 M and 0.1 M


Inoculate the yeast strain into 5 ml of liquid medium (2x YPAD or SC selection medium) and incubate overnight on a rotary shaker at 200 rpm and 30°C. Place a bottle of double strength YPAD broth (2x YPAD) and a 250 ml culture flask in the incubator as well.
===Boiled ssDNA===
This can be boiled once and used for many transformations. Re-boil after 4-6 freeze-thaws.
* Pre-heat a heat block to 100°C.
* Boil 1 mL of ssDNA for 5 min.
* Store at -20°C and keep on ice at all times during transformation.


'''Day 2'''
===50% PEG===
This can be stored at RT for a few days, or several weeks if in an airtight container such as a glass bottle (NOT Falcon tube). The following recipe is for 50 mL of PEG, which is good for ~200 transformations. Smaller amounts can be made by adjusting the amounts accordingly.


'''1.''' Determine the titer of the yeast culture by pipetting 10 ul of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10^6 cells/ml will give an OD600 of 0.1.  
* Measure out 25 grams of PEG 3500 into a 100 mL glass bottle.
* Add 25 mL water and vortex well. Let rest for 10 min.
** Note: PEG takes up volume when dissolved in water; this is why we start by adding only half the desired volume of water.
* Add water to 50 mL total volume. Vortex again and let rest until solution looks clear. Continue to vortex / let rest if necessary.
* Sterilize by autoclave on liquid 20 minute cycle or sterile filter into a new bottle.


===Transformation mix===
This mix should be made fresh every transformation. A good time to make this is after day 2 step 1, when you are waiting 3-5 hours for the cells to grow. First determine how much mix you need (the table below gives recipes for 1, 5, and 10 transformations), then add the following in order, and vortex until mixed:


'''2.''' Transfer 50 ml of the pre-warmed 2x YPAD to the pre-warmed culture flask and add 2.5 x 10^8 cells to give 5 x 10^6 cells/ml.
{|border="1" cellspacing="0" cellpadding="5" align="center"
|-
! rowspan=2 | Reagent
! colspan=3 | # reactions (amounts in uL)
|-
!1
!5 (6x)
!10 (11x)
|-
|PEG 3500 50% w/v:
|240
|1440
|2640
|-
|LiAc 1.0 M
|36
|216
|396
|-
|Boiled ssDNA (10 mg/ml)
|10
|60
|110
|-
|Plasmid DNA + dH2O (~1ug DNA)
|74
|444
|814
|-
!Total
!360
!2160
!3960
|}


==Day 1==


'''3.''' Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
'''1.''' Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C. Also incubate a flask of 50 mL YPD for every 10 transformations you will do.
Note:
i) It is important to allow the cells to complete at least two divisions.
ii)This will take 3 to 5 hours.
iii)This culture will give sufficient cells for 10 transformations.
iv) Transformation efficiency (transformants/ µg plasmid/108 cells) remains constant for 3 to 4 celldivisions.
Note: In practice, we dilute 0/N culture 1:100 and let it grow for 3-5 hours.


Checklist: ssDNA, PEG, LiAc (1M and 0.1M), icebox
==Day 2==
'''4.''' When the cell titer is at least 2 x 10^7 cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of 100mM LiAc.


'''1.''' Dilute the overnight culture 1:100 into fresh media, 50 mL per 10 transformations. Incubate the flask(s) for 3-5 hours at 30°C and 200 rpm.


'''5.''' Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.
'''2.''' When the cells are at OD ~ 0.3-0.5 (moderately cloudy to the eye), harvest the cells by pouring into 50 mL Falcon tubes and centrifuging at 3000 g for 5 min.
* It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.****


'''3.''' Wash the cells once in 25 mL of sterile water, by resuspending and centrifuging again, discarding the supernatant.


'''6.''' Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
'''4.''' Wash the cells once in 25 mL of 100mM LiAc.


'''7.''' Add 100 mM LiAc to a final volume of 500 ul.
'''5.''' Resuspend the cells in 1 mL of 100mM LiAc.
Note:If the cell titer of the culture is greater than 2 x 10^7 cells/ml the volume then increase the volume to maintain the titer of this suspension at 2 x 10^9 cells/ml. If the titer of the culture is less than 2 x 10^7 cells/ml then decrease volume.  


'''6.''' Transfer the cell suspension to a 1.5 mL microcentrifuge tube, centrifuge at 13000 rpm for 30 sec and discard the supernatant.


'''8.''' Vortex the cell suspension and pipette 50 µl samples (ca. 10^8 cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.
'''7.''' Add 100 mM LiAc to a final volume of 500 uL.


'''8.''' Vortex the cell suspension and aliquot 50 µl into 1.5 ml microfuge tubes, one for each transformation. Centrifuge at 13000 rpm for 30 sec and remove the supernatant.


'''9.''' Add the following reagents to each transformation in the order given:
'''9.''' Add 360 uL [[#Transformation mix|transformation mix]] to each tube and '''vortex for 30-60s to mix well'''. This last mixing is very important.


**PEG 3500 50% w/v:   240 µl
'''10.''' Incubate at 30°C for 30 min.
**LiAc 1.0 M: 36 µl
**Boiled SS-carrier DNA (2 mg/ml):   50 µl
**Plasmid DNA plus Water (use 0.1 - 10 mg tranforming DNA): 34 µl


'''11.''' Heat shock at 42°C for 15 min. (This time might vary depending on strain. Refer to the [[heat shock duration]] page for information about specific strains.)


'''10.''' Incubate for 30 minutes at 30°C
'''12.''' Centrifuge at 13000 rpm for 30 sec and remove the transformation reagents with a pipet (or aspirator).


Checklist: 42'C heating block or water bath
'''13.''' Pipette 1.0 ml of YPD into each tube; stir the pellet by with a pipet tip and vortex.
'''11.''' Incubate the tubes in a 42°C water bath for 20-25 min.
Note:The optimum time can vary for different yeast strains. Please test this if you need high efficiency from your transformations.  


'''14.''' Incubate at 30°C for 5 hours or O/N.


'''12.''' Microcentrifuge at top speed for 30 sec and remove the transformation reagents with a micropipettor.
'''15.''' Plate 200ul aliquots onto selective media. If cells have settled, mix by pipetting up and down before plating.


'''16.''' Incubate the plates at 30°C for 3 to 4 days and count transformant colonies.


'''13.''' Pipette 1.0 ml of YPD into each tube; stir the pellet by with a micropipette tip and vortex .
==References==
1. Note:We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants!!!!
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
 
 
'''14.''' Incubate at 30°C for approximately 5 hours, then plate 200ul aliquots onto selective plates.
 
 
'''15.''' Incubate the plates at 30°C for 3 to 4 days and count the number of transformants.
 
 
Modified from: Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.


http://home.cc.umanitoba.ca/~gietz/method.html
http://home.cc.umanitoba.ca/~gietz/method.html

Latest revision as of 07:49, 22 July 2011

Back to Springer Lab | Last updated by Jue Wang 10:53, 19 July 2011 (EDT)

YEAST TRANSFORMATION PROTOCOL

Note: This is a simplified (i.e. sloppier) version of a protocol by Gietz. If you need to troubleshoot, refer to the more detailed original version.

This procedure takes 4-6 hours from start to finish, plus an inoculation the day before.

Reagents

Besides culture media, the transformation requires the following reagents:

  • Icebox
  • Boiled salmon sperm DNA (ssDNA), 10 mg/mL
  • Polyethylene glycol (PEG) 3500, 50% w/v
  • Lithium acetate (LiAc), 1 M and 0.1 M

Boiled ssDNA

This can be boiled once and used for many transformations. Re-boil after 4-6 freeze-thaws.

  • Pre-heat a heat block to 100°C.
  • Boil 1 mL of ssDNA for 5 min.
  • Store at -20°C and keep on ice at all times during transformation.

50% PEG

This can be stored at RT for a few days, or several weeks if in an airtight container such as a glass bottle (NOT Falcon tube). The following recipe is for 50 mL of PEG, which is good for ~200 transformations. Smaller amounts can be made by adjusting the amounts accordingly.

  • Measure out 25 grams of PEG 3500 into a 100 mL glass bottle.
  • Add 25 mL water and vortex well. Let rest for 10 min.
    • Note: PEG takes up volume when dissolved in water; this is why we start by adding only half the desired volume of water.
  • Add water to 50 mL total volume. Vortex again and let rest until solution looks clear. Continue to vortex / let rest if necessary.
  • Sterilize by autoclave on liquid 20 minute cycle or sterile filter into a new bottle.

Transformation mix

This mix should be made fresh every transformation. A good time to make this is after day 2 step 1, when you are waiting 3-5 hours for the cells to grow. First determine how much mix you need (the table below gives recipes for 1, 5, and 10 transformations), then add the following in order, and vortex until mixed:

Reagent # reactions (amounts in uL)
1 5 (6x) 10 (11x)
PEG 3500 50% w/v: 240 1440 2640
LiAc 1.0 M 36 216 396
Boiled ssDNA (10 mg/ml) 10 60 110
Plasmid DNA + dH2O (~1ug DNA) 74 444 814
Total 360 2160 3960

Day 1

1. Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C. Also incubate a flask of 50 mL YPD for every 10 transformations you will do.

Day 2

1. Dilute the overnight culture 1:100 into fresh media, 50 mL per 10 transformations. Incubate the flask(s) for 3-5 hours at 30°C and 200 rpm.

2. When the cells are at OD ~ 0.3-0.5 (moderately cloudy to the eye), harvest the cells by pouring into 50 mL Falcon tubes and centrifuging at 3000 g for 5 min.

3. Wash the cells once in 25 mL of sterile water, by resuspending and centrifuging again, discarding the supernatant.

4. Wash the cells once in 25 mL of 100mM LiAc.

5. Resuspend the cells in 1 mL of 100mM LiAc.

6. Transfer the cell suspension to a 1.5 mL microcentrifuge tube, centrifuge at 13000 rpm for 30 sec and discard the supernatant.

7. Add 100 mM LiAc to a final volume of 500 uL.

8. Vortex the cell suspension and aliquot 50 µl into 1.5 ml microfuge tubes, one for each transformation. Centrifuge at 13000 rpm for 30 sec and remove the supernatant.

9. Add 360 uL transformation mix to each tube and vortex for 30-60s to mix well. This last mixing is very important.

10. Incubate at 30°C for 30 min.

11. Heat shock at 42°C for 15 min. (This time might vary depending on strain. Refer to the heat shock duration page for information about specific strains.)

12. Centrifuge at 13000 rpm for 30 sec and remove the transformation reagents with a pipet (or aspirator).

13. Pipette 1.0 ml of YPD into each tube; stir the pellet by with a pipet tip and vortex.

14. Incubate at 30°C for 5 hours or O/N.

15. Plate 200ul aliquots onto selective media. If cells have settled, mix by pipetting up and down before plating.

16. Incubate the plates at 30°C for 3 to 4 days and count transformant colonies.

References

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

http://home.cc.umanitoba.ca/~gietz/method.html

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