Chin-Sang Lab Volunteer Training
description
Transcript of Chin-Sang Lab Volunteer Training
January 2013
Chin-Sang Lab Volunteer Training
Overview
Basic Procedures
PCR, Digestions, Ligation, Transformation, Plasmid Prep, Egg Prep, Worm Lysis, Gel Electrophoresis, Gel Extraction & Clean Up Kits
Worm Care
Worm Picking Tips, Chunking, Gender Identifiers, Life Stages & Identifiers, Infected Plates
Crosses
Strain Notation, Cross Notation, Extrachromosomal & Integrated Strains, Example Problems
Overview (cont’d)
Lab Maintenance
Maintaining Sterility: Racking Tips, Retrieving Microcentrifuge Tubes, Sterile Technique, Autoclaving
General: Making Solutions, Dishwashing Procedure, Bleaching Culture Tubes
Next Steps
Tutorials, Checkpoints, Resources
Basic Procedures
The Big Picture
PCR
Digestion
LigationTransformatio
n
In All Procedures:
1. Know why you are doing the task
2. There are often many ways to perform the same task
Each procedure/kit has certain differences though, so this is why #1 is so important. By knowing the destiny of your product you can better select the method with which to make it.
3. Keep accurate and detailed notes of everything you do in the lab
PCR Basics
Used to amplify segments of DNA
Eg. Want to ligate that part into another plasmid so need more of it
Eg. Testing for the presence of a mutation
Mutant may amplify differently (eg. deletion mutation) or may have different cut sites (later shown in a digestion)
PCR
There are many different PCR kits used in the lab, each with its own attributes & protocol.
It’s important that you understand WHY you are performing the PCR (eg. is it for cloning?, to test for the presence of a gene?, etc.) That will help determine which polymerase to use
Keep notes of the PCRs you run and their purpose
PCR Polymerases
HiFi Polymerases
Able to proofread (contain exonucleases) fewer errors made∴
Used in cloning to have more accurate results
HiFi polymerases used in the lab: (will say HiFi on them)
Kapa HiFi
PCR Mastermix HiFi
PCR Polymerases
LowFi Polymerases
Not able to proofread
Used in testing for the presence of a gene mutation.
LowFi Polymerases used in the lab:
Taq Polymerase (made in the lab; protocol outlined here)
AccuStart II
PCR Reactions There are 3 basic steps that every PCR reaction will have:
Denaturation
Annealing
Extension
Here, we will outline the protocol to follow for the Taq polymerase the lab makes. Check the lab computer for the protocols for different kits.
ALWAYS CHECK THE PROTOCOL FOR THE RECIPE AND THE REACTION CONDITIONS FOR THE KIT YOU ARE USING!
PCR: Taq Protocol
Recipes
To adjust final volume, change amount of 10x PCR so the amount you add is 1/10 of the final volume and adjust ddH2O accordingly
To adjust the amount of DNA added, keeping the final volume the same, only adjust the amount of ddH2O added
Plasmid Genomic DNA Worm Lysis
DNA 1ul 5ul 10ul
10xPCR Buffer 3ul 3ul 3ul
Fwd Primer 2ul 2ul 2ul
Rev Primer 2ul 2ul 2ul
dNTPs 0.5ul 0.5ul 0.5ul
ddH2O 20.75ul 16.75ul 11.75ulTaq
Polymerase 0.75ul 0.75ul 0.75ul
Total Volume
30ul 30ul 30ul
Different amounts of DNA are used depending on what is being amplified
ddH2O is adjusted to account for the changes in
DNA volume
PCR (cont’d)
PCR Machine Settings
Most are preset
Temp Duration Purpose95°C 5 min Initial
Denaturation
95°C 30 sec Denaturation
Tm 15 sec Primer Annealing
72°C (varies) Varies based on polymerase
Extension
72°C 5 min Final Extension
To increase specificity of your PCR, increase the Tm
Polymerase Extension Times
•Taq: 1 min/kb•see sheets
x~30
PCR: Controls
Controls are important when troubleshooting failed reactions and to ensure that results obtained are valid.
PCR: Controls
Definition
Test Trial: the sample you are testing
Negative Control: should not yield any bands (test for contamination)
Positive Control: sample that will amplify our expected band. May not be possible for some PCR reactions.
PCR: Positive Controls
Positive controls can also be used to rule out other errors
Eg. Use 2 working primers to amplify another gene to rule out problems with reagents
Digestion Restriction enzymes cut DNA at palindromic sites unique to each
enzyme
Cutting makes sticky ends (in most cases). Complementary sticky ends can anneal in a ligation reaction
Recipe
Performing 1 digestion:
Can find protocol on Fermentas website
Google: Fermentas Fast Digest. Click “Complete L ist of F astD igest Restr iction E nzymes“. Select Enzyme. Click “Resources” Tab. Click “(Enzyme Name) Product Information”
Inactivate your enzyme with heat or purification.
Not all restriction enzymes are FD, although the lab mostly uses FD
enzymes.
Protocol for 1 Digestion
Plasmid PCR GenomicddH2O 15ul 16ul 30ul10x FD Buffer 2ul 3ul 5ul
DNA 1ul 10ul 10ulEnzyme 1ul 1ul 5ul
Total Volume 20ul 30ul 50ul
Incubation time @ 37°C 5 min 20 min 10 min•To digest with more than 1 enzyme, subtract the
amount of additional enzyme from the amount of ddH2O you add
Digesting More Than 1 Digestion (ie. multiple trials)
MasterMix
same incubation times
ddH2O 21.7ul
10x FD Buffer 3ul
DNA5ul (variable 1ul-10ul; depends on how
bright/concentrated the bands are; someone will tell you)
Enzyme 0.3ul-1ul
Total Volume 30ul
Ligation
Allows you to join together 2 fragments with complementary sticky ends into one piece of DNA
The overhangs of the 2 sticky ends will anneal together, so the 2 pieces being joined must have been cut with the same enzyme (or 2 enzymes that make complementary sticky ends) to create complementary overhangs
Different kits, with different attributes, can be used to obtain the same result
Fast Ligation & Overnight Ligation
same recipe proportions
different buffer & incubation times
Ligation
Recipe
5ul vector (Can be variable depending on concentration)
10ul insert (Can be variable depending on concentration)
4ul buffer (Fast Ligation: pre-aliquotted in “Ligation Buffers” box in the -20°C beside computer; Slow Ligase: in -20°C )
1ul T4 Ligase
Incubation Time
Fast: on bench (room temp), 15 min
Overnight: 15°C, overnight
Gibson Assembly
See NEBuilder for Gibson Assembly
Protocols:
http://nebuilder.neb.com
5’ 3’
5’ 3’
5’ 3’Pol +ligasePol +ligase
5’Pol +ligasePol +ligase
Chin-Sang LabGibson Assembly followed by PCR with outside primers
5’ 3’
5’Use this Gibslon assembly product as your PCR template with outside primersNote that the 5’ ends are “chewed” back. Normally this would be repaired with the vector backbone in a standard Gibson assembly reaction.
Two PCR fragments with overlapping sequences
Exonuclease chews back 5’ ends
DNA Pol fills in 3’ ends and Ligase joins
3’
Add some forward and reverse primer here?Say after 25 min? Will help fill in ends?Maybe inactivate Exonuclease first?
5’ 3’
5’Use this Gibson assembly product as your PCR template with outside primers
Forward primer
Reverse primer
5’
3’
5’Forward primerReverse primer
5’
3’
5’Forward primerReverse primer
5’5’
Forward primer
Reverse primer
The newly extended primer can serve as templates for future rounds of amplification of full length product
Primers extend
Denature
Heat Shock Transformation
1. Add 11ul of the ligation mixture to competent cells.
Competent cells are on the top shelf, right hand side of the -80°C in the hallway in a pitcher.
Cells must be kept cool for the “shock” to work. To do this, keep competent cells ①on ice at all times (unless otherwise indicated in procedure), allow the cells to ②thaw on ice rather than in your hand (gradual thawing ensures the cells aren’t harmed), use the cells immediately after removing them from the freezer.③
2. Keep mixture on ice for 15-20 min.
3. Heat shock at 42°C for 90 sec.
4. Put on ice for 1 min.
Transformations (cont’d)
5. Add 1ml 2xTY (use sterile technique).
6. Grow on shaker in the 37°C room for 45 min. Place plate with appropriate antibiotic resistance in the 37°C.
Plates found in clear 4°C fridge in the lab.
The plasmid backbone contains the antibiotic resistance gene
There are different antibiotic resistances that a plasmid can have (Ampicillin: plates are marked with a red line, Kanamycin: plates are marked with red/green line)
Giving bacteria that take up your plasmid antibiotic resistance allows you to select for the proper colony. Bacteria without the plasmid will not replicate when exposed to the antibiotic
Transformations (cont’d)
7. Plate out 100ul on the selection plate. Spin the rest of the cells down. Aspirate to 100ul.
8. Re-suspend the pellet and plate the remaining 100ul onto the plate from the 37°C room. Spread using ~5-10 beads. Place used beads in plastic bucket beside the sink.
9. Retrieve plates and look for colonies the next day.
Do not leave plates longer than overnight. Colonies will overgrow and combine. Joined colonies may not be identical genetically; individual colonies are. Come in the next day to remove the plate or ask someone in the lab to remove it for you.
Liquid Culture
Amplifies bacteria from selected colony in liquid media.
1. Pipette 3ml 2xTY + desired resistance into an autoclaved glass culture tube.
Growing the bacteria in liquid media that contains the antibiotic ensures that only the bacteria that keep the plasmid survive. If the challenge was removed, future generations in which the plasmid was rejected would grow alongside the bacteria with the plasmid of interest.
The antibiotic 2xTY is found in the clear 4℃ fridge.
2. Using a culture stick, pick one colony off the plate (try not to grab any agar) and swirl into media to remove bacteria from the tip.
3. Place the culture tube on the shaker in the 37°C room for ~16 hours. Do not leave it longer or it will overgrow.
Plasmid Prep/MiniPrep
Follow instructions in blue box that says “MiniPrep”
Box is on the shelf above where the worm plates are kept
Tips:
“Solution 1” is in the 4°C fridge
Diagnostic Digestion
Some undesired colonies may survive if during the ligation, the plasmid backbone closed without taking up the insert. Having a closed plasmid backbone will confer resistance for the cell that up took this plasmid (unless the insert is the resistance gene.)
A diagnostic digestion must be done to confirm that the surviving colony has the desired plasmid.
Diagnostic Digestion
2 methods:
1. Digest plasmid with same enzymes used to insert the insert (not ideal if the plasmid originally had an insert of a similar size).
2. Find a cut site unique to the insert.
How to make a composite part
1. Digest the plasmid and the insert with the same enzymes
2. Run both on a gel
3. Gel extract the desired bands (if needed)
4. Ligate the parts together
5. Transformation using the ligation product
6. Liquid culture
7. Miniprep
8. Diagnostic Digestion
Overview: How to Make a Composite Part
Example 1: Ligating insert into a multiple cloning site (MCS)
Diagnostic Digestion for Ex. 1
Because the section cut out of the original plasmid is only a few bp, digesting with the same 2 enzymes (EcoRI and KpnI) is a sufficient diagnostic.
Gel should reveal 2 bands, one should be the length of the plasmid backbone and the other should be the length of the insert
Overview: How to Make a Composite Part
Example 2: Switching one insert for another
Diagnostic Digestion for Ex. 2
GFP and RFP are approximately the same length. Re-digesting the ligated plasmid with the same 2 enzymes (Hindi III and Sma I) will not tell us anything. A plasmid that was initially undigested, a digested plasmid that RFP re-inserted into, and a correct plasmid that uptook the GFP will all appear the same on a gel.
To ensure you have the right plasmid, find a cut site unique to GFP.
Tip for Diagnostic Digestion
Don’t run your entire digestion on the gel. Save at least half of it in the -20°C in case something goes wrong with the gel. That way, you can quickly run a new gel instead of re-digesting your product.
Deactivating Enzymes
Check Fermentas protocol (same protocol used in digestion)
Some enzymes can be heat deactivated, others must be cleaned using the kit
2 general methods
1. Use the Enzymatic Clean-Up kit in the Blue Boxes
2. Heat the reaction in the PCR machines
Gel Electrophoresis
Separates bands of DNA based on their size
Altering the % agarose content of the gel and the voltage allow you to improve separation of the bands
**Always wear gloves when handling Ethidium Bromide (EtBr) and the gels after EtBr has been added. Only open EtBr in the fume hood. Dispose of EtBr pipette tips in the designated “EtBr Waste” bin.**
EtBr is found on the door of the fridge beside the computer
*The gel hardens quickly: once you pour the liquid gel in the casting tray put water in the beaker immediately so the remanence doesn’t solidify*
Gel Electrophoresis Protocol
Don’t run gel as long if using 2 rows of wells
If band is large, run longer. If band is small, run shorter.
Small Gel Large Gel
1x TBE 50 ml 100mlAgarose-Biotech
GradeDepends on %.
Usual 0.5 g (1%) Depends on %. Usual 1 g (1%)
EtBr 3ul 6ul
Voltage 100V 120V
Duration 60 min 60-75 min
Microwave 1 min & cool in fume hood
for ~2 min
Pour into gel casting tray & cool in fridge for at least 20 min
Gel Electrophoresis Tips
Variations
If separating bands of very similar size (eg. 200bp & 220 bp):
Increase gel %
Decrease voltage. Increases resolution. Must increase running time.
Tip
When mixing loading dye with DNA on Parafilm, place DNA on side of Parafilm previously covered with paper.
Gel Extraction
Allows you to remove a band from a gel (eg. when you are performing a ligation.)
To excise the band:
Bring the following to Ken Ko’s work room
UV glasses: in the drawer beside the sink
Razor: in the drawer beside the glasses & key
Microcentrifuge tubes: # bands excised=# tubes
Ladder Ruler
Gel Extraction (cont’d)
Place the gel with the gel tray on the UV box.
Put the glasses on, and turn the UV box on.
Do not look at the UV box without the glasses on
Keep the UV box on for as little as possible, because the UV light will damage the DNA
With the razor blade cut out your band of interest getting as little excess gel as possible.
Place the gel fragment in a microcentrifuge tube.
Ensure the UV box is turned off when you leave Walker’s lab
Gel Extraction (cont’d)
Removing the DNA from the gel
Follow instructions in blue box labelled “Gel Extraction”
Tips:
1. To measure weight of extracted gel, use scale in Bendena lab. Tare the scale using an empty microcentrifuge tube before weighing your sample.
2. To melt the gel place it in the 42℃ water bath for a longer time
Worm Lysis
Exposes DNA inside worms
Important for testing if worms are of a certain genotype
Followed by a PCR
Preparation:
Pour thermos of liquid N2
Have PicoFuge (mini centrifuge) beside the microscope where you are sitting
Gather enough PCR tubes for your samples & positive and negative controls
Determine which strain will be your positive control
Worm Lysis (cont’d) Protocol:
1. Each lysis sample contains 10ul. Mix 95% 1xPCR (if 10XPCR, dilute), 5% Proteinase K
2. Aliquot 10ul in the lid of each small PCR tube.
3. Pick the desired amount of worms.
4. Spin 2 at a time in PicoFuge.
5. Put tubes in liquid N2 immediately and leave them in there for a minimum of 10 min (longer is fine.)
6. Using the sieve beside the sink, pour the liquid N2 back into the large canister and catch the PCR tubes in the sieve.
7. Put the PCR tubes in the PCR machine under the “Worm Lysis” setting.
8. Include positive and negative controls. (Eg. +: lysis of strain with mutation of interest, -: lysis buffer)
Embryo Prep
Bleaching a plate of mixed stage worms kills all worms leaving only the embryos
Embryos left without food will arrest at L1
This protocol ensures all worms develop in synchrony by arresting all embryos in L1 before allowing them to proceed in their life cycle
Bleach Solution:
1mL 100% bleach
1mL 10M NaOH
8mL ddH2O
Embryo Prep (cont’d)
1. Add 1mL M9 to wash the plate.
2. Pipette M9 into microcentrifuge tube
3. Spin down in Picofuge
4. Aspirate
5. Add 1mL bleach solution
6. Turn tube for 90 sec to lyse worms (look at tube under microscope to see bodies dissolving)
7. Spin down
8. Aspirate
9. Add 1L bleach solution
Embryo Prep (cont’d)
10. Turn tubes for < 1 min (until clear)
11. Centrifuge at 8,000 rpm for 1 min
* Do not disturb the pellet-handle tube carefully*
12. Aspirate
*Do not aspirate the embryos*
13. Add 1mL M9
14. Repeat steps11-13 3-5 times
Check under microscope for embryos
Embryo Prep (cont’d)
15. Add 1mL M9
16. Put on shaker in the 20℃ incubator
17. Retrieve worms after 15 hours at the earliest
Worm Care
C. elegans Basics
Worms come in 3 genders:
Male , female
Hermaphrodite ⚥
It takes them about 3 days to become adults from the onset of embryogenesis
15°C: 5.5 days; 20°C: 3.5 days; 25°C: 2.5 days
Life span: 2-3 weeks (*If worms go into dauer they can remain there for ~3 months before proceeding onto L4*)
C. elegans Life Cycle
C. elegans can be grown from 16-25°C. Worms @ 25°C grow 2.1x faster than at 16°C. Worms @ 20°C grow 1.3x faster than worms at 16°C. Above stage
durations are at 22°C.
Life Stage Identification
Embryo
Laid EmbryoEmbryos in
Utero
Dauer
Non-aging state occurring after L1 due to unfavourable conditions
Subsequent life span is unaffected by dauer stage
Trigger:
Overcrowded
Starving
In temperatures too high (27°C)
Identification:
Long and skinny
Feeding ceases
Dauer (cont’d)
Behaviour:
On plate: lie motionless. Once disturbed, move rapidly.
Nictation: dauer-specific behaviour
the larva stands on its tail, waving its head in the air
Recovery:
occurs once favourable conditions are restored
within 1 hr of accessing food, the worm leaves dauer
QuickTime™ and aJVT/AVC Coding decompressorare needed to see this picture.
L4
L4 hermaphrodites are used in crosses
If they get too old they are able to fertilize themselves and are no longer useful in a cross
Gender Differences
Hermaphrodites
99.9% of the total population
Longer & fatter
Tail: Long and tapered
Sex chromosomes: XX
Males
0.1% of the total population
Slim and short
Tail: Fan extends from side
Sex chromosomes: X0
Making Males
wt males
Males are made by joining one gamete that contains an X chromosome and one that lacks sex chromosomes (0)
Gametes lacking sex chromosomes occur because of a nondisjunction event during gametogenesis
Offspring produced as a result of a mating event ( x ♂; as ⚥opposed to selfing) have a higher incidence of males ⚥because male sperm are 50:50 X:0
A line of non-mutant males can be maintained by taking 1 naturally occurring male and repeatedly crossing it into a N2 line
him mutants (High Incidence of Males)
him mutants (eg. him-5) interfere with the stable pairing of X chromosomes during meiosis, resulting in a higher frequency of nondisjunction events
him homozygous hermaphrodites will self to produce males at a 30% incidence (vs. 0.05% for N2 worms)
Identifying Males
Appearance
Smaller & skinnier than ⚥
Have a “fan” that extends from the tail
♂
Fan
Tail & Size Comparison: ♂ vs. ⚥
Fan
♂
⚥
Developing Fan
When performing crosses males should be picked before they’ve reached full maturity
An easy way to tell how mature a male is by his fan
Adult
L4
L3More severe
taper than the gradual
thinning in the ⚥
L4
L3
Adult
♂ ⚥
Male Tail: L3 & L4
Identifying Males
Behaviour
Males can often be found rubbing against hermaphrodites
Transferring Worms
When transferring worms keep the time that the plates are lidless to a minimum. This helps reduce the chances of the plate becoming contaminated.
Chunking
Used to move many worms (ie. hundreds) at once
Especially useful if plate is hard to pick from (ie. starved)
Only used to maintain homozygous lines (not heterozygous)
1. Remove sterilized spatula from EtOH & flame it
2. Cut through the agar of a populated plate using the spatula.
3. Lift the piece of agar onto the spatula and place it worm-side down onto a seeded plate. The worms will crawl out from under the chunk and onto the bacterial lawn on the new plate.
Worm Picking
Transfer one or a few worms at a time using a thin metal wire
2 Methods to Pick Worms:
Always flame pick before transferring a worm
1. Scooping the worm: Lower the tip of the wire and gently swipe the tip at the side of the worm and lift up.
2. Touch end of pick on OP50. Gently touch the back of a worm. Worm will stick to the bacteria.
Worm Picking (cont’d)
Putting the Worms Down:
Refocus the microscope on the edge of the bacterial lawn
Gently touch the surface of the agar and hold the pick there
The worm will crawl off towards the bacteria. For this reason it is best to place your pick very close to the lawn.
Worm Picking (cont’d)
Don’t leave the worm on the pick for too long or they will dry up & die
PRACTICE!
You will all be given your own plate of worms to maintain so you can practice picking. If you aren’t doing anything during your shift, practice picking until you have mastered it.
Contaminated Plates
Plates may become contaminated with mold, yeast, mites, or other bacteria
Cleaning the Stock
Chunk worms from an area away from the site of contamination onto a new plate. Place the chunk on the edge of the lawn.
Wait for the worms to crawl across the lawn before picking them onto another fresh plate.
Monitor the plate over the next few days to ensure that the contaminant did not also transfer
If the subsequent plate becomes contaminated repeat the procedure until the contaminant is removed
Contaminated Plates (cont’d)
Disposing of the Contaminated Plates
Most contaminated plates can be thrown directly in the autoclave bags
Wrap with Parafilm before throwing out if:
Mites on plate
Plate is badly contaminated
Crosses
Gene Notation
gene (allele)
eg. daf-2 (e979)
eg. daf-2 (e1370)
Allele Notation: The letter represented the lab that isolated the allele
Chin-Sang Lab: “qu”
Strain Notation
A strain can encompass many gene mutations, so using strain notation allows you to refer to a set of mutations in shorthand
DR 1979
Chin-Sang Lab: “IC”
•Lab that made the strain•All capitals
•Not italicized
•# assigned sequentially by lab as they register them
Microinjection
Makes transgenic worms
Inject DNA into the gonad
DNA is taken up into germ cell nuclei, forming extrachromosomal arrays
DNA can be delivered to many progeny in this way
∴ Microinject the mother’s gonad to see the trait in F1
Extrachromosomal Arrays
When DNA is microinjected it is not naturally incorporated into the worm’s genome; it remains extrachromosomal
Denoted by “Ex” in allele notation
Not all worms contain the DNA
eg. If the gene is GFP, some worms will not fluoresce, others will
Not all cells contain/can express the DNA. These worms are a genetic mosaic (different cells have different genotypes.)
eg. If the promoter dictates that GFP be expressed in all touch neurons, some may not fluoresce
Extrachromosomal Arrays
When crossing strains with extrachromosomal arrays:
Pick for the marker
eg. Only pick worms with GFP. Worms not expressing GFP do not have the DNA to express GFP.
The parent may or may not give the DNA to progeny
The parent NEVER gives the DNA to ALL the progeny; there will always be some that will not have the DNA.
Integrated Strain
The DNA has been integrated into a chromosome
Every worm in that strain contains the DNA
Every cell in the worm contains the DNA
Made by introducing DNA containing your gene of interest via microinjection and exposing the worms to mutagens
Mutations induce chromosome breaks
The extrachromosomal array you injected can be incorporated into the chromosome during DNA repair
C. elegans Chromosomes & Notation
5 pairs autosomes and one pair of sex chromosomes
Mutations occuring on different chromosomes are written in order
+ (I) ; + (II) ; unc-25::GFP (III) ; daf-18 (IV) ; him-5 (V) ; + (X)+ (I) ; + (II) ; daf-2 (III) ; + (IV) ; him-5 (V) ; + (X/0)
Chromosome #
•Wildtype•2 Normal
Chromosomes•wt chroms can
be omitted when writing
genotype
•Heterozygous•Both inherited chromosomes
contain a different mutation
•Heterozygous•1 inherited
chromosome=normal•1 inherited
chromosome=mutated
(I)(I)
(II)(II)
(IV)(IV)
(V)(V)
(III)(III)
++
++
unc-25::GFPdaf-
2+
daf-18
him-5
him-5
•Homozygous•Both inherited chromosomes
contain the same mutation
Sex Chromosome(s)
(X)(X/0)
++
Markers
When you are crossing in a “phenotype-less” gene you need to have a marker to show which progeny received the gene of interest
A segregation marker on the same chromosome as your gene of interest is used to follow how your gene is being segregated
The disappearance of the marker indicates the presence of the gene
Markers
eg. zdIs5 (I):
pmec-4::GFP
can be used as a segregation marker
pmec-4 is a promoter that is expressed in the touch neurons
pmec-4::GFP is a construct the lab made that fluoresces in the touch neurons. This provides a way to visualize the touch neurons.
Cross Basics
When setting up a cross pick 10 L3-L4 ♂ and 2 L4 ⚥
⚥ can be younger than L4 but NOT OLDER. past L4 start ⚥producing sperm and are able to fertilize themselves.
The 10:2 ratio ensures that the are preferentially fertilized by ⚥the males
It is imperative that you pick the correct genders.
In a cross, if even one is picked from the strain that was ⚥supposed to be the father, the predicted cross will not work.
In a selfing step, if a ♂ is picked instead of a no progeny will be ⚥produced
Set up a few trials (~3) of each cross in case something goes wrong on one of the plates.
Cross Example Background Info Desired end product:
Phenotypes:
rhIs-4: neuronal GFP (head)
oxis-12: seam-line GFP
daf-2 (III) daf-2 (III)
oxis-12 (X)
oxis-12 (X)
;
Cross ExamplerhIs-4 (III)
rhIs-4 (III)
him-5 (V)him-5 (V)
;♂ x him-5 (V)him-5 (V)
;
oxis-12 (X)
oxis-12 (X)
⚥P
rhIs-4 (III)
+ (III)
; him-5 (V)him-5 (V)
;oxis-12
(X)0
♂F1 1.0oxis-12 (X)
+ (X)
F1 are identical except ⚥for the sex chromosomes:
Generation
Probability
With him-5 there is a 30% chance of males. But, by
picking only males there is 100% chance this is the
genotype we are carrying into the next cross
daf-2 (III)daf-2 (III)
⚥
x
rhIs-4 (III)
daf-2 (III)
him-5 (V)+ (V)
oxis-12 (X)
+ (X)
;
;;
;1.0
+ (III) daf-2 (III)
him-5 (V)+ (V)
oxis-12 (X)
+ (X)
; ;The heads of other progeny
are NOT GREEN
∴ the probability of obtaining the desired genotype is 0.5. However, by picking only offspring with a green
head, there is 100% chance that this is genotype being used in the next
step.
⚥
⚥
Cross Example (cont’d)rhIs-4 (III)
daf-2 (III)
him-5 (V)+ (V)
oxis-12 (X)
+ (X)
; ;
Self
daf-2 (III) daf-2 (III)
oxis-12 (X)
oxis-12 (X)
;
rhIs-4 (III)
daf-2 (III)rhIs-4 (III)
rhIs-4 (III)
oxis-12 (X)
+ (X)
+ (X)+ (X)
Of the possible combinations for Chrom. III,
only the desired combination is NOT
GREEN.
SEGREGATION MARKER
NOT GREEN
BOTH GREEN
1.00.33
x = 0.33
⚥
Probability
Because there is only a 1/3 chance of getting the genotype we want from what we picked, we need to pick enough plates of single so ⚥that when they self, at least one will be homozygous for oxis-12.
A good number may be 12 plates of single . ⚥ Theoretically, we should see 4 plates of the desired genotype.
You need to OBTAIN RESULTS from the total number of plates you intended to or else the probability will be altered.
Eg. You pick 12 single plates and on 6 of the plates all the left ⚥the plate. Re-pick 6 single plates. If not, instead of working from a sample size of 12 with 1/3 chance of obtaining the correct genotype, you are now working from a sample size of 6 with the same odds. Although in theory you should still obtain 2 correct plates, the sample size is not big enough for this to be a likely outcome.
Cross Example (cont’d)
Of the 12 single plates, the correct plates will be those in which ALL the offspring have seam-line GFP (homozygous for oxis-12.)
CRISPR Cas9genome editing
Coming soon– still being optomised.
Just read about it here:
http://www.genetics.org/content/195/3/1181.full
http://www.genetics.org/content/195/3/635.full
Lab Maintenance
Maintaining a Sterile Environment
Autoclaving
Sterilized solutions and dry materials by creating a high P, high temp environment
Anything put in the autoclave must have autoclave tape put on it
Once in the autoclave, the tape will have black lines on it
Bottles
Autoclaved with loose caps
Flasks
Mouth covered in foil
Autoclave Settings
Dry Goods:
Normally autoclaved for 40 min with 15 min drying time
Gravity, Cycle 4
If you must combine dry and liquid things, dry good can go on a liquid cycle, HOWEVER, liquids can NEVER go on a dry cycle
The increased temperature and increased pressure quickly dissipate on a dry cycle. This causes the liquid to explode!
Autoclave Settings
Liquids:
<1L: 20 min cycle
4L: 30-50 min cycles
After autoclaving, label the bottle with its contents and the date
Removing Microcentrifuge Tubes
Reaching in with your hands can contaminate all the tubes
Instead,
Reach in with gloves; or
Shake the tubes out onto a paper towel. If more come out, tip them off the paper towel back into the stock.
Racking Pipette Tips
Rack tips with gloves on
Once pipette tip boxes are filled, close them with autoclave tape.
Sterile Technique
Prevents contaminants in the air from contaminating stock solutions
Best thing to do: minimize the time the lid is off the bottle
Flaming the bottle:
When you open a bottle, quickly touch the mouth of the bottle to the flame, followed by the lid
Do the same upon closing
Sterile Technique: 2xTY
2xTY is a bacterial growth media
Use gloves
EtOH the bench and the pipette before and after using 2xTY
Flame the bottle
General Lab Maintenance
Dishwashing Procedure
Flasks with live culture or contaminated solutions
Must be bleached before disposal in the sink
Other glassware
Wash with Alconox
Rinse 3x with hot water
Rinse 3x with distilled tap water (dH2O)
Culture Tubes
Bleaching Old Culture Tubes
Old culture tubes are stored on the top shelves until at least 1 rack has been filled
Pour the liquid into a beaker with bleach
Dispose of glass culture tube in glass container
Wash lids and place in buckets (drawers to the left of the sink) to be bleached
Culture Tubes
New Racks of Culture Tubes
Brand new culture tubes are used & topped with washed or new lids
Must be autoclaved on Dry Cycle 4 (autoclaved 40 minutes, with 15 minutes drying time)
Autoclaved tubes stored on lower shelf of the 2nd bench
Dilutions
Calculators:
http://www.endmemo.com/bio/dilution.php
http://www.restrictionmapper.org/dilutioncalc9.html
Worm Plates
Where the worms live
Make sure there are always at least ~300 plates
Plates are usually poured every two days
Worm Plates
Making Solution
3L aliquots
Made in 6L flask (liquid will expand in autoclave)
Follow recipe from book
Cover mouth of flask with tin foil and put a piece of autoclave tape on it
Autoclave on Liquid 2
Solution must be cooled to 65°C before adding in cholesterol (3ml), KPO4, CaCl2, MgCl2
Worm Plates: Preparation
Preparing the Plates
EtOH the counter where you place the plates and where you will be pouring
Remove small plates from bag and place in stacks 10 high
Have 300 plates ready
Worm Plates: Preparation
Preparing the PourBoy III
Sterilizing
Wipe the tubes with EtOH
Place the input tube in a 600mL beaker that is half filled with EtOH and warm water
Place the output tube in an empty 600mL beaker
Set the PourBoy III to Setting B and let the machine run continuously by setting the delay time to 0 sec.
Hold the tubes or they will tip the beakers over
Worm Plates: Preparation
Preparing for Clean-Up
Have a 600mL beaker half filled with water ready to microwave for when you are done pouring
Worm Plates: Preparation
To cool the flask:
Fill silver autoclave tin with cold water and place the flask inside
Swirl the flask every few minutes while cooling to ensure the agar doesn’t solidify on the edges
Once cooled, add the remaining, pre-aliquotted reagents
Worm Plates: Pouring
Set PourBoy III to either Setting B (11mL,) or Setting C (11mL, manual)
Empty the water from the autoclave tin and place the cooled flask in it, propped on an angle by a pipette tip box
Wipe the tubes with EtOH again and place the input tube at the bottom of the flask before wrapping the tin foil around the tube at the mouth of the flask to prevent contamination
Worm Plates: Pouring
Step on peddle to start pouring. Step on the peddle at any time to stop pouring
If on a manual setting, the peddle must be stepped on to start each new plate
Start from the bottom of the pile of plates, picking up the lid of the 10th plate and the other 9 plates above it
Once the plate is filled, place the stack on top and repeat for the 9th plate
If on automatic, stop the machine when changing stacks
Worm Plates: Clean-Up
With ~80 plates left place the clean-up beaker in the microwave for 5 min
Immediately once you are done pouring the plates, retrieve the beaker of hot water and start running it through the tube to remove any remnant agar
Use oven mitts when removing the water from the microwave
Fill the flask with water immediately after you are done pouring plates so the agar doesn’t solidify inside
If agar does solidify inside, remove it from the drain
Then wipe the tubes down with EtOH one last time
Worm Plates: Tips
The agar will solidify quickly in the flask, so once you start pouring, do the task from start to finish
*Do not let agar solidify in the PourBoy III*
Antibiotic Plates
Follow recipe in book for 1L portion
Antibiotics are in the freezer by the computer
Pour into larger plates (25ml/plate) using PourBoy III
Mark plates with the appropriate colour code:
Amp: red; Kan: green/red
Once the plates are dry (overnight) place them back in the clear bag that they came in
Tape the bag closed & write the antibiotic and the date the plates were poured on the tape
Store the bag of plates in the clear 4°C fridge
To Come...
Tutorials
Topics:
How to use AxioPlan
How to use Confocal Microscope
Searching the databases in the computer
If you’ve attended a tutorial it will be marked on the Volunteer Qualifications Chart so grad students can ask you to do higher level tasks as you become able to do them
Arrange a time with Tony
Take notes!
Resources for Additional Information
Lab Website:
https://qshare.queensu.ca/Users01/chinsang/www/chinsanglab/chin-sang_lab_manual.htm
WormBook
WormBase
Worm Atlas
Google!