Chin-Sang Lab Volunteer Training
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Chin-Sang Lab Volunteer Training January 2013 Overview ✤ Basic Procedures ✤ ✤ Worm Care ✤ ✤ PCR, Digestions, Ligation, Transformation, Plasmid Prep, Egg Prep, Worm Lysis, Gel Electrophoresis, Gel Extraction & Clean Up Kits 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 Ligation Transformation 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 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 11.75ul Taq 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 ✤ 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 PCR (cont’d) ✤ PCR Machine Settings ✤ x~30 Most are preset Temp 95°C 95°C Tm Duration 5 min 30 sec 15 sec Purpose 72°C (varies) Varies based on polymerase Extension 72°C 5 min Final Extension Initial Denaturation Denaturation Primer Annealing To increase specificity of your PCR, increase the Tm Polymerase Extension Times •Taq: 1 min/kb •see sheets 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 ✤ ✤ Not all restriction enzymes are FD, although the lab mostly uses FD enzymes. Google: Fermentas Fast Digest. Click “Complete List of FastDigest Restriction Enzymes“. Select Enzyme. Click “Resources” Tab. Click “(Enzyme Name) Product Information” Inactivate your enzyme with heat or purification. Protocol for 1 Digestion DNA Enzyme Plasmid 15ul 2ul 1ul 1ul PCR 16ul 3ul 10ul 1ul Genomic 30ul 5ul 10ul 5ul Total Volume 20ul 30ul 50ul Incubation time @ 37°C 5 min 20 min 10 min ddH2O 10x FD Buffer •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 ✤ ddH2O 21.7ul 10x FD Buffer 3ul DNA 5ul (variable 1ul-10ul; depends on how bright/concentrated the bands are; someone will tell you) Enzyme 0.3ul-1ul Total Volume 30ul same incubation times 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.co m Chin-Sang Lab Gibson Assembly followed by PCR with outside primers 5’ 3’ Two PCR fragments with overlapping sequences 5’ 3’ Pol +ligase 5’ 3’ Pol +ligase 5’ 3’ Exonuclease chews back 5’ ends DNA Pol fills in 3’ ends and Ligase joins 5’ Add some forward and reverse primer here? Say after 25 min? Will help fill in ends? Maybe inactivate Exonuclease first? 3’ 5’ Use this Gibslon assembly product as your PCR template with outside primers Note that the 5’ ends are “chewed” back. Normally this would be repaired with the vector backbone in a standard Gibson assembly reaction. 3’ Forward primer 5’ 5’ Reverse primer Use this Gibson assembly product as your PCR template with outside primers 3’ 5’ Reverse primer Forward primer 5’ Primers extend 3’ 5’ Reverse primer Forward primer 5’ Denature Forward primer 5’ 5’ Reverse primer The newly extended primer can serve as templates for future rounds of amplification of full length product 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, ① keep competent cells on ice at all times (unless otherwise indicated in procedure), ② allow the cells to thaw on ice rather than in your hand (gradual thawing ensures the cells aren’t harmed), ③ use the cells immediately after removing them from the freezer. ✤ 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 Small Gel Large Gel 1x TBE 50 ml 100ml Agarose-Biotech Grade Depends 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 ✤ Don’t run gel as long if using 2 rows of wells ✤ If band is large, run longer. If band is small, run shorter. 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 Embryos in Utero Laid Embryo 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 a JVT/AVC Coding decompressor are 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 ✤ Males ✤ 99.9% of the total population ✤ 0.1% of the total population ✤ Longer & fatter ✤ Slim and short ✤ Tail: Long and tapered ✤ Tail: Fan extends from side ✤ Sex chromosomes: XX ✤ 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 Adult L4 L4 L3 More severe taper than the gradual thinning in the ⚥ L3 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 •Lab that made the strain •All capitals •Not italicized ✤ •# assigned sequentially by lab as they register them Chin-Sang Lab: “IC” 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) ; + + (I) + (II) unc-25::GFP (III) daf-18 (IV) him-5 (V) + (X) (X) ; + (I) ; + (II) ; daf-2 (III) +(IV) (IV) him-5 ; him-5 (V) ; + + (I) + (II) (X/0) (V) + daf-2 (III) + (X/0) Chromosome # •Wildtype •2 Normal •Heterozygous •Both inherited Chromosomes chromosomes contain •wt chroms can be a different mutation omitted when writing genotype •Heterozygous •1 inherited chromosome=normal •1 inherited chromosome=mutated Sex Chromosome(s) •Homozygous •Both inherited chromosomes contain the same mutation 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 ⚥ ✤ ✤ ✤ The 10:2 ratio ensures that the ⚥ are preferentially fertilized by the males It is imperative that you pick the correct genders. ✤ ✤ ✤ ⚥ can be younger than L4 but NOT OLDER. ⚥ past L4 start producing sperm and are able to fertilize themselves. 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: daf-2 (III) ; oxis-12 (X) daf-2 (III) oxis-12 (X) ✤ Phenotypes: ✤ rhIs-4: neuronal GFP (head) ✤ oxis-12: seam-line GFP Cross Example Generation Probability ♂ rhIs-4 (III) ; him-5 (V) P rhIs-4 (III) F1 1.0 him-5 (V) x him-5 (V) ; oxis-12 (X) ⚥ him-5 (V) oxis-12 (X) ♂ rhIs-4 (III) ; him-5 (V) ; oxis-12 (X) + (III) him-5 (V) oxis-12 (X) + (X) 0 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 1.0 F1 ⚥ are identical except for the sex chromosomes: daf-2 (III) ⚥ daf-2 (III) x ∴ 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. The heads of other progeny are NOT GREEN rhIs-4 (III) ; him-5 (V) ; oxis-12 (X) daf-2 (III) + (V) + (X) ⚥ ; him-5 (V) ; oxis-12 (X) + (III) daf-2 (III) + (V) + (X) ⚥ Cross Example (cont’d) ⚥ rhIs-4 (III) ; him-5 (V) ; oxis-12 (X) daf-2 (III) + (V) + (X) Self Of the possible combinations for Chrom. III, only the desired combination is NOT GREEN. daf-2 (III) ; oxis-12 (X) daf-2 (III) oxis-12 (X) BOTH GREEN SEGREGATION MARKER rhIs-4 (III) daf-2 (III) oxis-12 (X) + (X) rhIs-4 (III) rhIs-4 (III) + (X) + (X) 1.0 x 0.33 NOT GREEN = 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 Cas9 genome 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 ✤ ✤ Bottles ✤ ✤ Once in the autoclave, the tape will have black lines on it 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/chi n-sang_lab_manual.htm ✤ WormBook ✤ WormBase ✤ Worm Atlas ✤ Google!
