Springer Lab: TransformationYeast

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(Day 2)
(YEAST TRANSFORMATION PROTOCOL)
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=YEAST TRANSFORMATION PROTOCOL=
=YEAST TRANSFORMATION PROTOCOL=
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'''Note:''' This is a simplified (i.e. sloppier) version of a protocol by Gietz. If you need to troubleshoot, refer to the more detailed version [http://home.cc.umanitoba.ca/~gietz/method.html here].
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'''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].
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This procedure takes 4-6 hours from start to finish, plus an inoculation the day before. Besides culture media, the transformation requires the following reagents, for which recipes and instructions can be found at the bottom of the page:
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This procedure takes 4-6 hours from start to finish, plus an inoculation the day before. Besides culture media, the transformation requires the following reagents:
* Icebox
* Icebox
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* Boiled salmon sperm DNA (ssDNA), 2 mL/mL ([[#Appendix: Preparing boiled ssDNA|instructions]])
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* Boiled salmon sperm DNA (ssDNA), 2 mL/mL
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* Polyethylene glycol (PEG) 3500, 50% w/v ([[Appendix: Preparing 50% PEG|instructions]])
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* Polyethylene glycol (PEG) 3500, 50% w/v
* Lithium acetate (LiAc), 1 M and 0.1 M
* Lithium acetate (LiAc), 1 M and 0.1 M
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==Day 1==
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==Preparing reagents==
 +
===Boiled ssDNA===
 +
* 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.
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Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C.
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===50% PEG===
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To make 4 mL of 50% PEG:
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==Day 2==
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* Measure out 2 grams of PEG 3500 into a 15 mL Falcon tube.
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* Add 2 mL water and vortex. Incubate for 5 min at 42°C and vortex again.
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'''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.
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** Note: PEG takes up a non-trivial volume when dissolved in water; this is why we start by adding only half the desired volume of water.
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* Add water to 4 mL total volume. Continue to heat / vortex until all PEG dissolves.
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'''2.''' Harvest the cells by pouring into 50 mL Falcon tubes and centrifuging at 3000 g for 5 min.
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* Store at RT for no more than a week.
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+
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'''3.''' Wash the cells once in 25 mL of sterile water, by resuspending and centrifuging again, discarding the supernatant.
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-
 
+
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'''4.''' Wash the cells once in 25 mL of 100mM LiAc.
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-
 
+
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'''5.''' Resuspend the cells in 1 mL of 100mM LiAc.
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+
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'''6.''' Transfer the cell suspension to a 1.5 mL microcentrifuge tube, centrifuge at 13000 rpm for 30 sec and discard the supernatant.
+
-
 
+
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'''7.''' Add 100 mM LiAc to a final volume of 500 uL.
+
-
 
+
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'''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.
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'''9.''' Add the following reagents to each transformation '''''in order''''':
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===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:
{|border="1" cellspacing="0" cellpadding="5" align="center"
{|border="1" cellspacing="0" cellpadding="5" align="center"
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|}
|}
 +
==Day 1==
 +
 +
Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C.
 +
 +
==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.''' 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.'''
'''11.''' Incubate at 42°C for 40 min.
'''11.''' Incubate at 42°C for 40 min.
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'''16.''' Incubate the plates at 30°C for 3 to 4 days and count transformant colonies.
'''16.''' Incubate the plates at 30°C for 3 to 4 days and count transformant colonies.
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==Appendix: Preparing boiled ssDNA==
 
-
 
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'''5.''' Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.
 
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* 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.****
 
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==Appendix: Preparing 50% PEG==
 
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.
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

Revision as of 10:37, 19 July 2011

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

Contents

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. Besides culture media, the transformation requires the following reagents:

  • Icebox
  • Boiled salmon sperm DNA (ssDNA), 2 mL/mL
  • Polyethylene glycol (PEG) 3500, 50% w/v
  • Lithium acetate (LiAc), 1 M and 0.1 M

Preparing reagents

Boiled ssDNA

  • 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

To make 4 mL of 50% PEG:

  • Measure out 2 grams of PEG 3500 into a 15 mL Falcon tube.
  • Add 2 mL water and vortex. Incubate for 5 min at 42°C and vortex again.
    • Note: PEG takes up a non-trivial volume when dissolved in water; this is why we start by adding only half the desired volume of water.
  • Add water to 4 mL total volume. Continue to heat / vortex until all PEG dissolves.
  • Store at RT for no more than a week.

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 SS-carrier DNA (2 mg/ml) 50 300 550
Plasmid DNA plus Water (use 0.1 - 10 ug tranforming DNA) 34 204 374
Total 350 2160 3960

Day 1

Inoculate the yeast strain into 5 ml YPD and incubate overnight on a rotary shaker at 200 rpm and 30°C.

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. 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. 11. Incubate at 42°C for 40 min.

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 approximately 5 hours (or dilute into 5 mL YPD O/N).

15. Plate 200ul aliquots onto selective media.

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

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

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