McClean: Colony PCR (Yeast): Difference between revisions

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==Protocol==
==Protocol==


Add approximately 0.6μL of cells (tiny amount) with the tip of toothpick into the bottom of PCR tubes.
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 for 1min (2X)
 
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===
===PCR Reaction Mix===
Line 35: Line 39:
| HotMaster Taq DNA polymerase ||0.5μL
| HotMaster Taq DNA polymerase ||0.5μL
|-
|-
| Sterile Water|| 40.5μL
| Sterile Water|| 41.5μL
|-
|-
| style="background:#f0f0f0;"| Total Volume
| style="background:#f0f0f0;"| Total Volume
Line 41: Line 45:
|}
|}


==''Day 1''==
===PCR Program===
# 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.
Run PCR on Colony PCR program :
*95°C 4min
*For the following steps, reduce the temperature ‐0.5°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 5 min
**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.
 
 
*'''[[User:Taylor D. Scott|Taylor D. Scott]] 20:32, 19 March 2016 (EDT)''': I had success with the following program adapted from the touchdown protocol and the HotMaster manual (see references below). The amplification is lower (as is expected with fewer cycles), but it might work if you just need to quickly verify a colony.
 
*95°C 4 min
*Repeat the following 30X
**94°C 30 sec
**52°C 30 sec (use the Applied Biosystems calculator [see references below] to find the right annealing temperature)
**65°C 1 min 30 sec (approximately 1 min/kb)
*72°C 5 min
*4°C hold
 
==='Colony PCR with the cheap Taq'===
Add approximately 1/4 of a colony to a PCR tube and microwave for 30 sec. Immediately put the tubes on ice after microwaving.
To each tube add the following:
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10X buffer ||10 μL
|-
| 10mM dNTP mix || 2 μL
|-
| 10μM Primer 1|| 1 μL
|-
| 10μM Primer 2 || 1 μL
|-
| Taq DNA polymerase || 2 μL
|-
| Autoclaved milliQ water || 84 μL
|-
| style="background:#f0f0f0;"| Total Volume
|  style="background:#f0f0f0;"| 100 μL
|}
 
Mix by pipetting up and down.


==''Day 2''==
Run PCR with the following program:
# 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.
# Grow in shaking incubator for about 3-5 hours.
# Turn on 42°C water-bath (for heat-shock) if it is not already on.
# 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.
# 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.
# 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.
# Add 300 μL T mix to each eppendorf tube of cells.  Per one transformation reaction add '''IN ORDER''':
#*240 μL 50% PEG 3350
#*35 μL 1.0 M lithium acetate
#*25 μL 2 mg/ml sssDNA
#*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)
# Vortex to resuspend cells.
# Incubate for 30 minutes at 30°C.
# 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.
# 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.)
# 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.
# 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:
#* Plate 150 µl of sterile water and add 20 µl cell suspension in one selection plate #1.
#* Plate the remaining 180 µl in selection plate #2
#Incubate at 30 °C.  Colonies should appear after 2-4 days.


==Recovery step for drug resistance markers and 5-FOA==
*94°C 5 min
*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 them.  This is done after you have removed the transformation mix but ''before'' you plate the cells.  You have two options for recovery:
*Repeat the following 25X
# 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.
**94°C 30 sec
# 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.
**T<sub>a</sub> 30 sec
**72°C 1 min/kb
*72°C 5 min
*4°C hold


*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.
Where T<sub>a</sub> is the appropriate annealing temperature for your primers. Gradient PCR can be used if the calculated T<sub>a</sub> is not working.
 
==='Agarose Gel Electrophoresis'===
* Find stock gel or prepare the agarose +ethidium bromide gel according to these instructions: [[McClean: Agarose for gels]]
** Ethidium bromide is a known carcinogen. Use gloves, don't inhale fume, follow good lab practice.
* Melt the gel in the microwave and let it cool until it is warm but not painfully hot to the touch.
* Fill the gel container with the agarose gel until it just begins to overflow out of the small clear plastic chamber into the large container.
**[[Image:Gel_Container.jpg|||100px|||]]
*Let the gel dry
* Place your gel in the electrophoresis container and ensure that the TAE solution is filled to the marked line. If not, find TAE solution in the stock room and dilute it to 1x solution and fill the container.
** The DNA will have a net negative charge, and so ensure that the holes in the gel face the anode (-) so that the DNA will flow to the cathode (+).
**[[Image:Gel_Electrophoresis_MM.jpg|||100px|||]]
* Insert 10 microL of DNA ladder (your reference points) into a well. For reference, 1kb ladder mix has 0.5kb, 1kb, 1.5kb, 2kb 3kb (double bright), 4kb, 5kb, 6kb, 8kb, 10kb fragments of DNA.
*On parafilm, pour ~1 microL drops of "10x blue juice" found in the "Gel Supplies Wisc Mini Fridge."
**[[Image:Juice_on_Parafilm.jpg|||100px|||]]
** Add 10 microL of your PCR product to the drops, pipetting up and down to mix them.
** Pipet your PCR product+ blue juice into each well. It is denser than the salt solution, and will sink into the hole.
* Press "Set" on the device so that the two LEDs next to "Volt" are lit, and then press the "Run" button. (You may need to set a maximum amount of time too)
* A ~500bp fragment of DNA will require ~45min to be properly separated. A 7kb fragment of DNA will require ~1 1/2 hrs to be distinguishable.
*Place your gel slabs in the UV chamber, turn the UV chamber on, take a picture, turn on the connection to the printer, turn on the printer, then print the image by selecting Set> Print on the camera.
**[[Image:uv_chamber.jpg|||100px|||]]
*Save the picture in you lab notebook and document the significance of each column.
**[[Image:Lab_Notebook_Gel_Electrophoresis.jpg|||100px|||]]
 
==='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==
==Notes==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
Please feel free to post comments, questions, or improvements to this protocol.  
#List troubleshooting tips here.   
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
 
#Anecdotal observations that might be of use to others can also be posted here.   
'''*[[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.
 
'''*[[User:Megan N McClean|Megan N McClean]]''' 15:36, 25 March 2013 (EDT) See [http://openwetware.org/wiki/McClean:_Sequencing_Colony_PCR_Product Sequencing Colony PCR Product] for how I prep my colony PCR products for sequencing


Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
*'''[[User:Taylor D. Scott|Taylor D. Scott]] 13:51, 15 April 2016 (EDT)''':The cheap Taq protocol can probably be scaled back to 50 μL per reaction, but I haven't tried it.


==References==
==References==
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS
Botstein Lab protocols: http://www.princeton.edu/genomics/botstein/protocols/colony_PCR.htm
CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
 
HotMaster manual: https://www.5prime.com/media/3388/hotmaster%20taq%20dna%20polymerase%20manual_5prime_1044359_032007.pdf
 
Applied Biosystems Tm calculator: http://www6.appliedbiosystems.com/support/techtools/calc/


==Contact==
==Contact==
*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 20 July 2011 (EDT)'''
'''[[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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[[Category:Yeast]]


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Latest revision as of 10:51, 15 April 2016


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 41.5μL
Total Volume 50μL

PCR Program

Run PCR on Colony PCR program :

  • 95°C 4min
  • For the following steps, reduce the temperature ‐0.5°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 5 min
    • 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.


  • Taylor D. Scott 20:32, 19 March 2016 (EDT): I had success with the following program adapted from the touchdown protocol and the HotMaster manual (see references below). The amplification is lower (as is expected with fewer cycles), but it might work if you just need to quickly verify a colony.
  • 95°C 4 min
  • Repeat the following 30X
    • 94°C 30 sec
    • 52°C 30 sec (use the Applied Biosystems calculator [see references below] to find the right annealing temperature)
    • 65°C 1 min 30 sec (approximately 1 min/kb)
  • 72°C 5 min
  • 4°C hold

'Colony PCR with the cheap Taq'

Add approximately 1/4 of a colony to a PCR tube and microwave for 30 sec. Immediately put the tubes on ice after microwaving. To each tube add the following:

Reagent Volume
10X buffer 10 μL
10mM dNTP mix 2 μL
10μM Primer 1 1 μL
10μM Primer 2 1 μL
Taq DNA polymerase 2 μL
Autoclaved milliQ water 84 μL
Total Volume 100 μL

Mix by pipetting up and down.

Run PCR with the following program:

  • 94°C 5 min
  • Repeat the following 25X
    • 94°C 30 sec
    • Ta 30 sec
    • 72°C 1 min/kb
  • 72°C 5 min
  • 4°C hold

Where Ta is the appropriate annealing temperature for your primers. Gradient PCR can be used if the calculated Ta is not working.

'Agarose Gel Electrophoresis'

  • Find stock gel or prepare the agarose +ethidium bromide gel according to these instructions: McClean: Agarose for gels
    • Ethidium bromide is a known carcinogen. Use gloves, don't inhale fume, follow good lab practice.
  • Melt the gel in the microwave and let it cool until it is warm but not painfully hot to the touch.
  • Fill the gel container with the agarose gel until it just begins to overflow out of the small clear plastic chamber into the large container.
  • Let the gel dry
  • Place your gel in the electrophoresis container and ensure that the TAE solution is filled to the marked line. If not, find TAE solution in the stock room and dilute it to 1x solution and fill the container.
    • The DNA will have a net negative charge, and so ensure that the holes in the gel face the anode (-) so that the DNA will flow to the cathode (+).
  • Insert 10 microL of DNA ladder (your reference points) into a well. For reference, 1kb ladder mix has 0.5kb, 1kb, 1.5kb, 2kb 3kb (double bright), 4kb, 5kb, 6kb, 8kb, 10kb fragments of DNA.
  • On parafilm, pour ~1 microL drops of "10x blue juice" found in the "Gel Supplies Wisc Mini Fridge."
    • Add 10 microL of your PCR product to the drops, pipetting up and down to mix them.
    • Pipet your PCR product+ blue juice into each well. It is denser than the salt solution, and will sink into the hole.
  • Press "Set" on the device so that the two LEDs next to "Volt" are lit, and then press the "Run" button. (You may need to set a maximum amount of time too)
  • A ~500bp fragment of DNA will require ~45min to be properly separated. A 7kb fragment of DNA will require ~1 1/2 hrs to be distinguishable.
  • Place your gel slabs in the UV chamber, turn the UV chamber on, take a picture, turn on the connection to the printer, turn on the printer, then print the image by selecting Set> Print on the camera.
  • Save the picture in you lab notebook and document the significance of each column.

'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.

*Megan N McClean 15:36, 25 March 2013 (EDT) See Sequencing Colony PCR Product for how I prep my colony PCR products for sequencing

  • Taylor D. Scott 13:51, 15 April 2016 (EDT):The cheap Taq protocol can probably be scaled back to 50 μL per reaction, but I haven't tried it.

References

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

HotMaster manual: https://www.5prime.com/media/3388/hotmaster%20taq%20dna%20polymerase%20manual_5prime_1044359_032007.pdf

Applied Biosystems Tm calculator: http://www6.appliedbiosystems.com/support/techtools/calc/

Contact

Megan N McClean

or instead, discuss this protocol.


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