McClean: Colony PCR (Yeast)

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(New page: <!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --> ==Overview== Our lab's version of the Geitz lithium-acetate transformation method. ==Materials== * 1M LiAC * 100mM LiAC...)
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==Overview==
==Overview==
 +
Our lab's version of yeast colony PCR, adapted from the Botstein Lab's protocol.  Generally, we use this protocol for checking transformations (ie, to check that a drug marker or fluorescent protein has inserted into the genome correctly) or for PCRing up a piece of DNA from the genome to send for sequencing.
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Our lab's version of the Geitz lithium-acetate transformation method.
+
==Materials==
 +
*HotMaster Taq Polymerase
 +
*10x HotMaster Buffer with Mg<sup>2+</sup>
 +
**The polymerase and buffer come in the 5 Prime kit FP220320 ordered from Fisher
 +
*10mM dNTPs
 +
*Forward primer (10μM)
 +
*Reverse primer (10μM)
 +
*Sterile H<sub>2</sub>O
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==Materials==  
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==Protocol==
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* 1M LiAC
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Add approximately 0.6μL of cells (tiny amount) with the tip of a sterile toothpick into the bottom of PCR tubes or plate.  Once you've put cells into the PCR vessel, put the end of the toothpick into ~100μL sterile YPD in either an eppendorf or the well of a culture plate. You will use this to inoculate an overnight culture if your colony PCR works.  Keep the eppendorfs or culture plate at either room temperature or 30°C while you run the PCR, either is fine. 
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* 100mM LiAC
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* 50% w/v PEG MW 3350 (Sigma P3640)
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* Sterile H20
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* Single-stranded carrier DNA (Sigma D1626, 2.0 mg/ml in TE buffer pH 8.0)
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* Appropriate selective plates (SC-URA, YPD+G418, etc)
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* Sterile 4mm glass-beads for plating (Fisher 11-312B)
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==Stock Solutions==
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Microwave cells in the PCR tube/plate for 1min (2X).  Put microwaved cells on ice. 
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'''Polyethylene glycol PEG 50% w/v (Sigma P3640)'''
 
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* Make up 50% w/v with H<sub>2</sub>O and filter-sterilize with a 0.45uM filter unit (Nalgene 295-4545 or similar).  It will take a long time for the PEG to work it's way through the filter, be patientWe don't autoclave the PEG.  Store in a tightly capped container to avoid evaporation.  
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Add the reaction mix (described below) to the PCR tube/plate.  It is recommended to make up a master mix if you are doing multiple coloniesPut the PCR tubes/plate into the thermocycler and run the Colony PCR program described below.
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'''Single-stranded carrier DNA (Sigma D1626)'''
+
===PCR Reaction Mix===
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# Weight out 200mg of the DNA into 100ml of TE buffer.  Disperse the Dna into solution by drawing it p and dwn repeatedly in a 10-ml pipette.  Mix vigorosly on a magnetic stirrer for 2-3 hours or until fully dissolved.  Alternatively, leave the covered solution mixing at this stage overnight in a cold rom.
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# Aliquot the DNA into 100μL portions and store at -20°C. 
+
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# Prior to use, the aliquot should be boiled and then quickly cooled on ice.  We use a thermocycler to heat the DNA to 95°C for 25 minutes and then rapidly cool it on ice.
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Once the salmon sperm has been boiled it can be freeze-thawed 3 or 4 times before transformation efficiencies begin to decrease. In practice, we boil the DNA before every transformation.
 
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'''TE Buffer (pH 8.0)'''
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{| {{table}}
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*10 mM Tris-HCL (pH 8.0)
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| align="center" style="background:#f0f0f0;"|'''Reagent'''
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*1.0 mM EDTA
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| align="center" style="background:#f0f0f0;"|'''Volume'''
 +
|-
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| 10x HotMaster Taq Buffer with Mg2+ ||5μL
 +
|-
 +
| 10mM dNTP mix ||1μL
 +
|-
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| 10μM Primer 1|| 1μL
 +
|-
 +
| 10μM Primer 2 ||1μL
 +
|-
 +
| HotMaster Taq DNA polymerase ||0.5μL
 +
|-
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| Sterile Water|| 40.5μL
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|-
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| style="background:#f0f0f0;"| Total Volume
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|  style="background:#f0f0f0;"| 50μL
 +
|}
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'''1.0M Lithium acetate stock solution (LiAc)'''
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===PCR Program===
 +
Run PCR on Colony PCR program :
 +
*95°C 4min
 +
*For the following steps, reduce the temperature ‐1°C each cycle and cycle 30x's
 +
**94°C 1min
 +
**65°C 1min
 +
**68°C 2min
 +
*For the following steps, cycle 30x's
 +
**94°C 30sec
 +
**50°C 30sec
 +
**72°C 1min
 +
*72°C 5min
 +
*4°C Forever
 +
**Please don't leave the thermocycler running at 4°C for longer than an hour or so, it wears out the machine. If you need to leave your PCR for longer, please change the last step of the program so that instead of holding at 4°C the program just ends (letting the samples come to room temperature).  Letting the sample come to room temperature, even overnight, does not seem to cause any problems for the DNA.
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* Prepare as a 1.0 M stock in distilled deionized H<sub>2</sub>O; filter-sterilize.  The final pH should be between 8.4 and 8.9
 
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'''Glass-beads for plating (Fisher 11-312B)'''
 
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*Pour beads into a small glass bottle (typically wide-mouthed 100ml or 250ml bottles work well) and autoclave on a 15 minute dry cycle to sterilize
 
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==''Day 1''==
 
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# Inoculate the strain to transform from a single colony into 5mls of YPD in a test tube.  Put on the roller drum at 30°C overnight.
 
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==''Day 2''==
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==='Freezing Down Positive Transformants'===
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# Inoculate 50 ml of YPD with 500 μL of the YPD overnight culture in a 250 ml flask.  The 500 µl volume is approximate, and depends on the density of the strain you inoculate.
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Once you have run the colony PCR and confirmed which colonies are correct and which are not, take the 100μL of YPD that you set aside before, inoculate it into 4mls of fresh YPD in a test tube, and grow it to saturation overnightUse this to freeze down glycerol stocks of the strain the following morning.
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# Grow in shaking incubator for about 3-5 hours.
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# Turn on 42°C water-bath (for heat-shock) if it is not already on.
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# Harvest the cells by centrifuging in Eppendorf centrifuge model 5810R at 4000rpm (3130 xg) for 5 min.  Resuspend pellet in 25 ml of sterile water by vortexing briefly.  Pellet again and then resuspend in 1 ml of 100 mM LiAc.
+
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# Transfer cell suspension to a 1.5 ml eppendorf tube, centrifuge at 3,000 xg for 2 min in an Eppendorf 5418 centrifuge and discard supernatant by removing it with a pipette.
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# Add 400 µl 100 mM LiAc and resuspend cells by pipetting up and down.  Aliquot 50 μL into 1.5 ml tubes (1 for each transformation).  Pellet cells (3,000 xg for 2 min) and remove supernatant by aspiration.
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# Add 300 μL T mix to each eppendorf tube of cells.  Per one transformation reaction add '''IN ORDER''':
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#*240 μL 50% PEG 3350
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#*35 μL 1.0 M lithium acetate
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#*25 μL 2 mg/ml sssDNA
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#*50 μL  sterile H20 and 20 μL of DNA (Note: You are aiming for a final concentration between 0.1-10 μg for plasmid DNA.  Adjust your DNA and water amounts to add 70 μL of volume total)
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# Vortex to resuspend cells.
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# Incubate for 30 minutes at 30°C.
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# Incubate tubes in a water bath at 42°C for 20-25 (up to 40) min.  The time may need to optimized for your strain and transformation conditions.
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# Microfuge at 3,000 xg for 15s, and remove transformation mix with a micropipette.  (NOTE: If you are transforming cells with a drug resistance marker such as KanMX, NatMX, HygMX or selecting for 5-FOA resistance, '''DO NOT''' plate your cells now, you need to do a recovery step.  See below.)
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# Add 200 µL of sterile water to each tube and resuspend cells by pipetting it up and down as gently as possible if high efficiency is important.
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# Plate your cells using glass beads to spread the cells.  Add 3-4 glass beads to each plate that you will be using, add about 200μL of cells + water, and spread by shaking the plate horizontally.  To ensure single colonies:
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#* Plate 150 µl of sterile water and add 20 µl cell suspension in one selection plate #1.
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#* Plate the remaining 180 µl in selection plate #2
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#Incubate at 30 °C.  Colonies should appear after 2-4 days.
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-
 
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==Recovery step for drug resistance markers and 5-FOA==
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*If you are plating your cells onto plates with G418, clonNat, hygromycin, or 5-FOA (basically if you are trying to select for anything BUT ability to grow without a particular amino acid) you need to give your cells some time to recover and express the resistance marker after you've transformed themThis is done after you have removed the transformation mix but ''before'' you plate the cells.  You have two options for recovery:
+
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# Gently resuspend cells in 1ml of YPD.  Put this tube at 30°C for 1-4 hours (with a tube clamp to keep the eppendorf from popping open).  After recovery, spin down the cells, resuspend in sterile water, and plate as above.
+
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# Gently resuspend cells in 1ml of YPD.  Plate 200μL cuture onto a YPD plate.  In the morning, replica plate this lawn of cells onto a selection plate.  You should see colonies in 2-4 days.  We usually save the rest of the cells resuspended in YPD overnight at 30°C (with a tube clamp to keep the eppendorf from popping open) and plate about 200μL of this onto a selection plate in the morning just as a back-up.
+
-
 
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*We have found that plating onto YPD and replica plating the next day gives the best results.  For 5-FOA transformations it seems crucial to do it this way, as allowing the cells to grow in liquid YPD for any amount of time allows cells with mutations in URA3 to arise and these are able to grow on 5-FOA.
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==Notes==
==Notes==
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Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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Please feel free to post comments, questions, or improvements to this protocol.  
-
#List troubleshooting tips here. 
+
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#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
+
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#Anecdotal observations that might be of use to others can also be posted here. 
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-
 
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
 +
 +
'''*[[User:Megan N McClean|Megan N McClean]]''' The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly.  The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone's time to figure out a shorter program.  Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.
 +
 +
'''*[[User:Megan N McClean|Megan N McClean]]''' We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions.  If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.
==References==
==References==
-
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS
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Botstein Lab protocols: http://www.princeton.edu/genomics/botstein/protocols/colony_PCR.htm
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CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
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==Contact==
==Contact==
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*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 20 July 2011 (EDT)'''
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'''[[User:Megan N McClean|Megan N McClean]] '''
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
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Revision as of 12:10, 4 October 2011


Contents

Overview

Our lab's version of yeast colony PCR, adapted from the Botstein Lab's protocol. Generally, we use this protocol for checking transformations (ie, to check that a drug marker or fluorescent protein has inserted into the genome correctly) or for PCRing up a piece of DNA from the genome to send for sequencing.

Materials

  • HotMaster Taq Polymerase
  • 10x HotMaster Buffer with Mg2+
    • The polymerase and buffer come in the 5 Prime kit FP220320 ordered from Fisher
  • 10mM dNTPs
  • Forward primer (10μM)
  • Reverse primer (10μM)
  • Sterile H2O

Protocol

Add approximately 0.6μL of cells (tiny amount) with the tip of a sterile toothpick into the bottom of PCR tubes or plate. Once you've put cells into the PCR vessel, put the end of the toothpick into ~100μL sterile YPD in either an eppendorf or the well of a culture plate. You will use this to inoculate an overnight culture if your colony PCR works. Keep the eppendorfs or culture plate at either room temperature or 30°C while you run the PCR, either is fine.

Microwave cells in the PCR tube/plate for 1min (2X). Put microwaved cells on ice.


Add the reaction mix (described below) to the PCR tube/plate. It is recommended to make up a master mix if you are doing multiple colonies. Put the PCR tubes/plate into the thermocycler and run the Colony PCR program described below.

PCR Reaction Mix

Reagent Volume
10x HotMaster Taq Buffer with Mg2+ 5μL
10mM dNTP mix 1μL
10μM Primer 1 1μL
10μM Primer 2 1μL
HotMaster Taq DNA polymerase 0.5μL
Sterile Water 40.5μL
Total Volume 50μL

PCR Program

Run PCR on Colony PCR program :

  • 95°C 4min
  • For the following steps, reduce the temperature ‐1°C each cycle and cycle 30x's
    • 94°C 1min
    • 65°C 1min
    • 68°C 2min
  • For the following steps, cycle 30x's
    • 94°C 30sec
    • 50°C 30sec
    • 72°C 1min
  • 72°C 5min
  • 4°C Forever
    • Please don't leave the thermocycler running at 4°C for longer than an hour or so, it wears out the machine. If you need to leave your PCR for longer, please change the last step of the program so that instead of holding at 4°C the program just ends (letting the samples come to room temperature). Letting the sample come to room temperature, even overnight, does not seem to cause any problems for the DNA.



'Freezing Down Positive Transformants'

Once you have run the colony PCR and confirmed which colonies are correct and which are not, take the 100μL of YPD that you set aside before, inoculate it into 4mls of fresh YPD in a test tube, and grow it to saturation overnight. Use this to freeze down glycerol stocks of the strain the following morning.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

*Megan N McClean The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly. The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone's time to figure out a shorter program. Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.

*Megan N McClean We probaby don't need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and doing smaller reaction volumes.

References

Botstein Lab protocols: http://www.princeton.edu/genomics/botstein/protocols/colony_PCR.htm

Contact

Megan N McClean

or instead, discuss this protocol.

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