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Chin-Sang Lab Volunteer Training

Chin-Sang Lab Volunteer Training. January 2013. Overview. Basic Procedures PCR, Digestions, Ligation, Transformation, Plasmid Prep, Egg Prep, Worm Lysis, Gel Electrophoresis, Gel Extraction & Clean Up Kits Worm Care

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Chin-Sang Lab Volunteer Training

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  1. Chin-Sang Lab Volunteer Training January 2013

  2. 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

  3. 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

  4. Basic Procedures

  5. The Big Picture PCR Digestion Ligation Transformation

  6. 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

  7. 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)

  8. 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

  9. 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

  10. 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

  11. 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!

  12. 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 Different amounts of DNA are used depending on what is being amplified ddH2O is adjusted to account for the changes in DNA volume

  13. PCR (cont’d) • PCR Machine Settings • Most are preset To increase specificity of your PCR, increase the Tm x~30 • Polymerase Extension Times • Taq: 1 min/kb • see sheets

  14. PCR: Controls • Controls are important when troubleshooting failed reactions and to ensure that results obtained are valid.

  15. 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.

  16. 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

  17. 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 List of FastDigest Restriction Enzymes“. 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.

  18. Protocol for 1 Digestion • To digest with more than 1 enzyme, subtract the amount of additional enzyme from the amount of ddH2O you add

  19. Digesting More Than 1 Digestion (ie. multiple trials) • MasterMix • same incubation times

  20. 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

  21. 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

  22. Gibson Assembly See NEBuilder for Gibson Assembly Protocols: http://nebuilder.neb.com

  23. Chin-Sang Lab Gibson Assembly followed by PCR with outside primers 5’ 3’ Two PCR fragments with overlapping sequences 5’ 3’ Exonuclease chews back 5’ ends Pol +ligase DNA Pol fills in 3’ ends and Ligase joins 5’ 3’ 5’ Pol +ligase Add some forward and reverse primer here? Say after 25 min? Will help fill in ends? Maybe inactivate Exonuclease first? 3’ 5’ 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.

  24. Forward primer 3’ 5’ 5’ Reverse primer Use this Gibson assembly product as your PCR template with outside primers 3’ 5’ Forward primer Reverse primer 5’ Primers extend 3’ 5’ Forward primer Reverse 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

  25. 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.

  26. 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

  27. 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.

  28. 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.

  29. 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

  30. 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.

  31. 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

  32. Overview: How to Make a Composite Part Example 1: Ligating insert into a multiple cloning site (MCS)

  33. 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

  34. Overview: How to Make a Composite Part Example 2: Switching one insert for another

  35. 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.

  36. 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.

  37. 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

  38. 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*

  39. 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. Microwave 1 min & cool in fume hood for ~2 min Pour into gel casting tray & cool in fridge for at least 20 min

  40. 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.

  41. 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

  42. 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

  43. 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

  44. 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

  45. 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)

  46. 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

  47. 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

  48. 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

  49. Embryo Prep (cont’d) • 15. Add 1mL M9 • 16. Put on shaker in the 20℃ incubator • 17. Retrieve worms after 15 hoursat the earliest

  50. Worm Care

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