McClean: Tetrad Dissection: Difference between revisions

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==Overview==
==Overview==
This protocol describes dissection of yeast tetrads.  In our lab, we primarily use tetrad dissection for constructing strains for genetic and biochemical experiments.


Our lab's version of the Geitz lithium-acetate transformation method.
An accomplished yeast biologist can dissect a plate of spores in 20 minutes; for a beginner, 2 hours is not unusual.  Tetrad dissection is a learnable skill. Your initial attempts will likely be frustrating.  If you persevere, you will be richly rewarded by your new ability to wield one of the most powerful tools in the yeast geneticist’s toolbox.  When you first learn to do tetrad dissection, make sure to ask for help from someone in the lab who is experienced at doing it!  It really helps to have someone with a practiced eye point out what a well-digested culture looks like, what a tetrad looks like under the dissecting scope, etc.


==Materials==  
==Materials==  
* β-glucuronidase (Sigma G7770, stored in 4°C refrigerator)
* Sporulated yeast culture
* Sterile Water
* "Dry" YPD dissection plates
**Everyone has a particular way that they like their plates for tetrad dissection.  You are basically aiming for ''dry'' and ''level''.  To make dissection plates, add 25 ml YPD with a plastic strippette to plates on a very level surface.  Once solid, invert.  Let dry at room temperature for ~3 days.  Bag.  These plates are best after aging for a while.


* 1M LiAC
* 100mM LiAC
* 50% w/v PEG MW 3350 (Sigma P3640)
* Sterile H20
* Single-stranded carrier DNA (Sigma D1626, 2.0 mg/ml in TE buffer pH 8.0)
* Appropriate selective plates (SC-URA, YPD+G418, etc)
* Sterile 4mm glass-beads for plating (Fisher 11-312B)


==Stock Solutions==
==Stock Solutions==


'''Polyethylene glycol PEG 50% w/v (Sigma P3640)'''
'''Stock Solution 1'''
 
* This is a very simple solution, so we only need a one line description of how to make it.  
* 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 patient.  We don't autoclave the PEG.  Store in a tightly capped container to avoid evaporation.   
 
'''Single-stranded carrier DNA (Sigma D1626)'''
# 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.
# Aliquot the DNA into 100μL portions and store at -20°C. 
# 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.
 
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.
 
'''TE Buffer (pH 8.0)'''
*10 mM Tris-HCL (pH 8.0)
*1.0 mM EDTA
 
'''1.0M Lithium acetate stock solution (LiAc)'''
 
* 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
 
'''Glass-beads for plating (Fisher 11-312B)'''
*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


==''Day 1''==
'''Stock Solution 2'''
# 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.


==''Day 2''==
This is a more involved solution, so we will describe how to make it in several steps:
# 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.
# Step 1  
# Grow in shaking incubator for about 3-5 hours.
# Step 2
# Turn on 42°C water-bath (for heat-shock) if it is not already on.
# Step 3
# 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==
==Protocol==
*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:
# Step 1
# 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.
# Step 2
# 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.
# Step 3


*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.


==Notes==
==Notes==
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
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. Happy to have your input!
#List troubleshooting tips here.   
#List troubleshooting tips here.   
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==Contact==
==Contact==
<!--Change the information below to your info if you add a new protocol-->
*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 20 July 2011 (EDT)'''
*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 20 July 2011 (EDT)'''


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Revision as of 11:10, 30 September 2011


Overview

This protocol describes dissection of yeast tetrads. In our lab, we primarily use tetrad dissection for constructing strains for genetic and biochemical experiments.

An accomplished yeast biologist can dissect a plate of spores in 20 minutes; for a beginner, 2 hours is not unusual. Tetrad dissection is a learnable skill. Your initial attempts will likely be frustrating. If you persevere, you will be richly rewarded by your new ability to wield one of the most powerful tools in the yeast geneticist’s toolbox. When you first learn to do tetrad dissection, make sure to ask for help from someone in the lab who is experienced at doing it! It really helps to have someone with a practiced eye point out what a well-digested culture looks like, what a tetrad looks like under the dissecting scope, etc.

Materials

  • β-glucuronidase (Sigma G7770, stored in 4°C refrigerator)
  • Sporulated yeast culture
  • Sterile Water
  • "Dry" YPD dissection plates
    • Everyone has a particular way that they like their plates for tetrad dissection. You are basically aiming for dry and level. To make dissection plates, add 25 ml YPD with a plastic strippette to plates on a very level surface. Once solid, invert. Let dry at room temperature for ~3 days. Bag. These plates are best after aging for a while.


Stock Solutions

Stock Solution 1

  • This is a very simple solution, so we only need a one line description of how to make it.

Stock Solution 2

This is a more involved solution, so we will describe how to make it in several steps:

  1. Step 1
  2. Step 2
  3. Step 3

Protocol

  1. Step 1
  2. Step 2
  3. Step 3


Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

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.

Contact

or instead, discuss this protocol.


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