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Notebook



Protocols


Yeast Transformation

Written up by Bowman Clark

Purpose

This is a protocol for the transformation of plasmids into yeast.


Materials

  • Yeast culture
  • 1.5 mL sterile tube
  • Pipette
  • One-Step buffer
  • 10 microliters DTT - 1g - $42.90 Sigma Aldrich
  • 5 microliters ssDNA - 1mg - $32 Sigma Aldrich
  • 3 microliters plasmid
  • Sterile water

One-Step buffer - adding 80 microliters of (Possibly divide by factor of 10)
  • 2.06g LiAc - 250 g = $51.50
  • 40g PEG
  • Make 80 mL solution in water

      • Equipment
      • 8000 rpm microcentrifuge – 134
      • 42°C incubator - Water Bath B025
      • vortex – 134

      Protocol

      1. Start a 5 mL yeast culture and grow overnight
      2. Take 500 μl of overnight culture and place in 1.5 mL sterile tube. Label tube with strain name and plasmid name
      3. Spin in microcentrifuge for 30 sec at 8K rpm to pellet cells
      4. Remove and discard supernatant
      5. Resuspend pellet in 1 mL sterile water by gently pipetting up and down
      6. Pellet the cells and centrifuge at 8000 rpm for 30 sec again
      7. Remove and discard supernatant
      8. Heat ssDNA at 90-100°C for 5 min
      9. Add 80 μl One-Step buffer (vortex after each addition), 1 μl of 1M DTT, 5 μl of ssDNA, and 5 μl of plasmid. Mix well
      10. Incubate at 42°C for 30-60 min
      11. Add 1 mL sterile water to transformation, mix and spin at 8000 rpm for 30 seconds
      12. Remove 800 μl liquid from tube and resuspend transformed cells in residual liquid
      13. Plate on selective media
      14. Parafilm plates and allow to grow for 2-4 days

      Discussion

      1. Add more plasmid if transformations are not working
      2. Make a new 1-step buffer for every transformation - DO NOT USE AN OLD STOCK

      Source

      Nina Serratore from the Briggs Lab
      Others: Clontech Yeast Protocol Handbook, http://www.bio-protocol.org/e96 Kiran Aslam, Hansen 235 (Hazbun Lab)

      Paragraph form

      Inoculate 5 mL of yeast broth and grow the culture up overnight. Pipette 500 μL of the culture into a sterilized 1.5 mL tube. Label the tube. Centrifuge the tube for 30 seconds at 8000 rpm to pellet the cells. Discard the supernatant. Add 1mL of sterile water to the tube and resuspend the cells by gently pipetting up and down. Centrifuge the tube again for 30 seconds at 8000 rpm to pellet the cells. Discard the supernatant. Add the following and vortex after each addition: 80 μL One-step buffer, 10 μL of 1 M DTT, 5 μL of ssDNA, and 3 μL of plasmid. Mix well. Incubate the tube at 42 degrees C for 30 minutes. Add 1 mL of sterile water to the transformation and mix. Centrifuge again at 8000 rpm for 30 seconds. Remove and discard 800 μL of liquid from the tube, and resuspend the cells in the remaining liquid. Plate on selective media.



      Ligation Protocol

      written by Bowman Clark

      Purpose

      Protocol for preparing Parts for assembly


      Materials

      • 21 ul of digested plasmid
      • 15 ul of each digested part
      • 24 ul ddH20
      • 9 ul 10x T4 Ligase Buffer
      • 6 ul T4 Ligase
      • PCR Tubes

      Equipment

      • Thermocycler (BIND 222)
      • Water Bath (BIND 134)

      Protocol
      1. Add appropriate amounts of vector, insert, and water to PCR tubes according to the table below
      2. Heat tubes for 2 minutes in water bath at 42 C to free up sticky ends
      3. Pipette 10x buffer up and down and add 1.5 ul to each tube
      4. Pipette T4 ligase up and down and add 1 ul to each tube, pipetting up and down in each tube after the addition of the T4
      5. Incubate at 16 C for 16 hours in thermocycler

      Ligation Table

      Item:
      		 1:06 1:03 1:01 3:01 6:01 1:00
      Vector (ul)
      		 .7 1.4 3 6 6 3
      Insert Total (ul)
      		 8.4 8.4 6 4 2 0
      dH2O (ul)
      		 2.4 1.7 3.5 2.5 4.5 9.5
      10x Buffer (ul)
      		 1.5 1.5 1.5 1.5 1.5 1.5
      T4 Ligase (ul)
      		 1 1 1 1 1 1
      Total (ul)
      		 15 15 15 15 15 15

      Discussion

      Video explanation: https://vimeo.com/45842813

      Source


      Protocol Paragraph Form:

      First, add the appropriate amount of vector, insert and water to PCR tubes for desired ligation ratios. Next, heat the tubes in a water bath at 42 C to free up the sticky ends. Pipette the T4 Ligase Buffer and add 1.5 ul toT4 each tube. Then pipette the T4 Ligase up and down and add 1 ul to each tube pipetting the mixture up and down after addition. Finally incubate the tubes at 16 C for 16 hours in the thermocycler.



      Restriction Digest Protocol

      Written up by Bowman Clark (Adjusted by Lexi Petrucciani)

      Purpose

      To prepare parts for assembly

      Materials

      • Ice and bucket/container
      • Eppendorf Tubes
      • Part A (Purified DNA, > 16ng/ul)
      • Part B (Purified DNA, > 16ng/ul)
      • Linearized plasmid backbone (25ng/ul)
      • dH2O
      • NEB Buffer 2
      • BSA
      • Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DpnI
      • Thermal cycler or Thermomixer

      Equipment

      List all the equipment needed to complete this protocol, including what lab # in Bindley or another building it can be found in

      Protocol

      1. Keep all enzymes and buffers used on ice.
      2. Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes, and flick/spin them to collect the liquid at the bottom of the tube.
      3. Add 500ng of DNA to the appropriately labelled tube. Add distilled water to the tubes for a total volume of 36.5ul in each tube.
        1. Calculation example (with 25ng/ul as DNA sample concentration):
          500ng ÷ 25ng/ul = 20ul of DNA sample
          36.5ul (total volume) – 20ul (DNA sample) = 16.5ul of distilled water
      4. Pipet 5ul of NEB Buffer 2 to each tube.
      5. Pipet 0.5ul of BSA to each tube.
      6. Add 4uL of the appropriate enzymes
        1. For 3A:
          1. Part 1: EcoRI and SpeI
          2. Part 2: XbaI and PstI
          3. Backbone: EcoRI and PstI(IF NOT ALREADY LINEARIZED)
        2. For Standard:
          1. Insert: EcoRI and SpeI
          2. Vector: EcoRI and XbaI
      7. The total volume in each tube should be 50ul. Mix well by vortexing. Spin the samples briefly to collect all of the mixture to the bottom of the tube.
      8. Incubate the restriction digests at 37°C for 4 hours, then use immediately or store at -20C.

      3A Assembly

      Item:
      		 Part A: Part B: Backbone:
      DNA
      		 500ng 500ng 500ng
      dH2O
      		 Adjust to 36.5ul Adjust to 36.5ul Adjust to 36.5ul
      NEB Buffer 2
      		 5ul 5ul 5ul
      BSA
      		 .5ul .5ul .5ul
      Enzyme 1
      		 4ul EcoRI 4ul XbaI 4ul EcoRI
      Enzyme 2
      		 4ul SpeI 4ul PstI 4ul PstI

      Discussion

      IF THE BACKBONE IS ALREADY LINEARIZED THEN YOU DON’T NEED TO DIGEST IT.

      Video explanation: https://vimeo.com/45793760

      Source


      Protocol Paragraph Form

      3A Digest

      Add 500 ng of DNA to an Eppendorf Tube. Add dH20 to 36.5 ul. Add 5ul NEB Buffer 2, 0.5 ul BSA, and 4 ul of each enzyme. Vortex, then incubate at 37C for 4 hours.


      Experiment Log

      Experiment:
      Name(s)
      Date
      Miniprep of yeast backbones from E. coli culture (pRS313, pRS315, pRS316) Everyone (and the high schoolers) 6/15/2015
      Preparation of competent E. coli Mark, Lexi 6/19/2015
      Transformation of backbones into yeast culture Tony, Bo 6/22/2015
      3A Assembly of killswitch promoter to RBS Everyone 6/23/2015
      Transformation of GFP into E. coli to test competency of cells we made ourselves Bo, Lexi 6/24/2015
      Digestion of promoter, rbs, lysing agent, and terminator Bo, Lexi, Kate 6/24/2015
      Ligation and transformation of promoter rbs lysing agent and terminator Bo, Lexi, Kate 6/25/2015
      PCR of G-Block Fragments Mark, Melissa, Erich, Suraj, Jill 6/25/2015
      //2015
      Resuspension and Dilution of G-Blocks and Primers Mark, Melissa, Erich, Suraj, Jill 6/25/2015
      PCR (3 replicates) of LiP1 and LiP2 Mark, Melissa, Erich, Suraj, Jill 6/26/2015
      PCR (3 replicates) of Lac1 and Lac2 Mark, Melissa, Erich, Jill 6/26/2015
      Run a gel to check digestion of promoter rbs lysing agent and terminator Bo, Lexi, Kate 6/26/2015
      PCR (3 replicates) of MnP1 and MnP2 Mark, Melissa, Jill 6/29/2015
      PCR (3 replicates) of AKR1, VP1, VP2 Melissa 6/30/2015
      PCR (3 replicates) of AKR2, Tyr1, Tyr2 Melissa, Erich 7/1/2015
      Gel of Lac1, Lac2, MnP1, MnP2, VP1 Mark, Jill 7/1/2015
      PCR (3 replicates) of all gBlocks Mark, Melissa, Jill, Erich 7/1/2015
      Miniprep of promoter, rbs, lysing agent, and terminator Bo, Lexi, Kate 7/6/2015
      Digestion of promoter with rbs and lysing agent, with terminator Bo, Lexi, Kate 7/7/2015
      PCR Purification (of MnP1&2, AKR1&2, VP1&2, and Tyr1) Suraj, Melissa 7/7/2015
      PCR of all G-Block Fragments Jill, Mark, Erich 7/7/2015
      Inoculation of liquid yeast cultures Mark 7/7/2015
      LB Agar with Amp plates Bo, Lexi, Kate 7/8/2015
      Gel of LiP1, LiP2, Lac1, Lac2, Tyr2 Mark, Melissa, Erich, Suraj, Jill 7/8/2015
      Gibson Assembly with PCR purified gBlocks (Mn1+Mn2, AKR1+AKR2, VP1+VP2) Mark 7/8/2015
      Gel of LiP1, LiP2, Lac1, Lac2, Tyr2 (of second PCR attempt) Mark, Melissa, Erich, Suraj, Jill 7/8/2015
      PCR Purification of LiP1 and LiP2 Jill, Erich, Melissa, Suraj 7/8/2015
      Gibson Assembly with PCR purified gBlocks (LiP1 + LiP2) Jill, Erich, Melissa, Suraj 7/8/2015
      E. Coli Transformation of Promoter-RBS and LysingAgent-Terminator Ligations Bo, Lexi, Kate 7/8/2015
      Yeast Transformation of backbones pRS313, pRS315, and pRS316 Mark, Tony 7/8/2015
      Gel of Gibson Results LiP, MnP, AKR, VP Erich, Suraj, Melissa 7/9/2015
      LB broth with amp Bo, Lexi, Kate 7/9/2015
      Ligation of promoter+rbs and lysing agent+terminator Bo, Lexi, Kate 7/9/2015
      E. Coli Transformation of Promoter-RBS and LysingAgent-Terminator Ligations Bo, Lexi, Kate 7/10/2015
      Gibson Assembly of PCR purified VP1 + VP2 Jill, Erich, Melissa, Suraj 7/10/2015
      Gel of Gibson Results VP Jill, Erich, Melissa, Suraj 7/10/2015
      Ethanol Precipitation of VP1 and VP2 Jill, Erich, Melissa, Suraj, Mark 7/10/2015
      Non-inoculated yeast plates test Tony 7/10/2015
      Inoculation of Promoter+RBS and LysingAgent+Terminator E. Coli Bo, Lexi, Kate 7/12/2015
      Preparation of Yeast glycerol stocks for long-term storage Mark, Erich 7/13/2015
      Miniprep of promoter+rbs and lysing agent+terminator Bo, Lexi, Kate 7/13/2015
      Gibson Assembly of Ethanol Precipitated VP1+VP2 Jill, Erich, Melissa, Suraj 7/13/2015
      Gel of Gibson Results VP Jill, Erich, Melissa, Suraj 7/13/2015
      Inoculation of liquid yeast cultures Mark 7/13/2015
      Second inoculation of liquid yeast cultures for competent cell preparation Mark 7/14/2015
      Transformation of second diluted yeast cultures with pRS313 Mark, Jill, Erich, Melissa, Suraj 7/14/2015
      Inoculation of liquid yeast cultures Mark 7/14/2015
      Dilution (10x, 25x, 50x, 100x) of inoculated yeast cultures Mark 7/15/2015
      Miniprep of promoter+rbs and lysing agent+terminator Bo, Lexi, Kate 7/15/2015
      Digestion of promoter+rbs and lysing agent+terminator Bo, Lexi, Kate 7/15/2015
      Preparation of E. Coli glycerol stocks for P+R and L+T Bo, Lexi, Kate 7/15/2015
      Overlap PCR of MnP1 and 2 (w/ primers and w/o) Mark, Jill, Erich, Melissa, Suraj 7/15/2015
      Gel of PCR Overlap Results Jill, Erich, Melissa, Suraj 7/15/2015
      Ligation of promoter+rbs+lysing agent+terminator Bo, Lexi, Kate 7/15/2015
      Transformation of promoter+rbs+lysing agent+terminator Bo, Kate, Lexi 7/16/2015
      Gel Electrophoresis of MnP Overlap PCR Mark, Suraj 7/16/2015
      Overlap PCR of LiP (straight gBlocks and PCR amplified gBlocks) Mark, Suraj 7/16/2015
      Gel Extraction of MnP Erich 7/16/2015
      Overlap PCR of LiP (straight gBlocks) Suraj, Tony, Arren, Jill, James 7/16/2015
      Gel of Overlap PCR of LiP Suraj, Jill, Melissa, Erich 7/17/2015
      PCR Amplification of Lac1, Lac2, Tyr2 Suraj, Jill, Melissa 7/20/2015
      Overlap PCR Part 2 of MnP Erich 7/20/2015
      Overlap PCR (with calculated Tm) of VP Suraj, Jill, Melissa 7/20/2015
      Ligation of promoter+rbs+lysing agent+terminator Bo, Lexi, Kate 7/20/2015
      Overlap PCR (with calculated Tm) of AKR Suraj, Melissa, Erich 7/21/2015
      Restriction Enzyme Digest and Ligation of MnP to pRS315 Mark, Erich 7/21/2015
      LB w/ chlor made Bo, Kate, Lexi 7/21/2015
      Inoculation of full device and E. coli Bo, Kate, Lexi 7/21/2015
      PCR Amplification of LiP1, LiP2, Lac1, Lac2, VP1, VP2, AKR1, AKR2, Tyr1, Tyr2 Melissa, Jill 7/21/2015
      E. coli transformation of MnP + pRS315 Mark 7/22/2015
      Gel of PCR Products (LiP1, LiP2, Lac1, Lac2, VP1, VP2, AKR1, AKR2, Tyr1, Tyr2) Suraj, Erich 7/22/2015
      Gibson assembly of AKR1 + AKR 2 Jill 7/22/2015
      Gel of Gibson Results AKR Suraj 7/22/2015
      PCR Purification of AKR2, LiP2, VP1, VP2 Jill 7/22/2015
      Mini prep + nanodrop Bo, Lexi, Kate 7/23/2015
      Gibson Assembly of VP1 + VP2 Mark 7/23/2015
      Gel Extraction of LiP1, Lac1, Lac2, AKR1, Suraj, Melissa, Jill 7/23/2015
      Gel Tyr1, Tyr2, Lac2, Gibson Assembley of AKR Suraj, Tony 7/23/2015
      Streaking of new Yeast (BY4741) plate Mark 7/23/2015
      EcoRI and PstI digest of AKR and MnP Mark 7/27/2015
      Liquid Inoculation of Yeast MST WT Cultures Erich 7/27/2015
      Digestion of GFP and full device for insertion into chlor and amp backbone Lexi, Kate 7/27/2015
      PCR of Lac2 and Gibson AKR Tony, Melissa 7/27/2015
      Gel electrophoresis of Lac2 (for extraction), AKR(test), & Tyr1(for extraction) Tony, Melissa 7/27/2015
      Vector Insertion of AKR and MnP into pSCB13C Mark 7/27/2015
      Transformation of AKR and MnP and GFP Bo, Kate, Lexi 7/28/2015
      Yeast Transformation of backbones pRS313, pRS315, and pRS316 Erich 7/28/2015
      LB + Chlor plates made Tony 7/28/2015
      Gel of Lac1 and Lac2 PCR amplified products (140uL in big wells) Suraj, Melissa 7/29/2015
      PCR Purification of LiP Gibson Results Jill, Melissa 7/29/2015
      Digestion of Full Device and yeast backbone Bo, Lexi, Kate 8/3/2015
      Digest of Mnp, AKR, GFP, full device and backbone Bo, Kate, Lexi 8/3/2015
      EtBr Gel of (Lane1: Ladder, Lane2: Tyr Gibson 20uL, Lane3: PCRLac1 A 10uL, Lane4: Lac1B, Lane5: Lac1C, Lane6: Lac2A, Lane7: Lac2B, Lane8: Lac2C) Jill, Erich, Suraj, Melissa 8/3/2015
      Transformation of AKR, MnP, GFP, and full device Bo, Kate, Lexi 8/4/2015
      Gel of Lac1 and Lac2 PCR amplified products (140uL in big wells) (Lane 1: Ladder, Lane 2: Lac1, Lane3: Lac2) Jill, Erich, Suraj, Melissa 8/4/2015
      Gel of (Lane1: Ladder, Lane2: 5ul Tyr 1 Gel extract, Lane3: 5uL Tyr2 Gel Extract) Jill, Erich, Suraj, Melissa 8/4/2015
      Gel Extraction of PCR amplified Lac1 and Lac2 Jill, Suraj, Erich 8/4/2015
      Gibson Assembly of Tyr1+Tyr2 (different gel extraction than before) Jill, Melissa 8/4/2015
      Gibson Assembly of Lac1 + Lac2 gel extracts Jill, Melissa 8/5/2015
      Gel of Gibson Assembly results (Lane1: Ladder, Lane2: Tyr, Lane3: Lac) Jill, Suraj, Melissa 8/5/2015
      Gibson Assembly of Tyr1+2 and Lac1+2 (with more DNA added) Jill, Suraj, Melissa 8/5/2015
      Gel Extract of Gibson Assembly Results Tyr Erich 8/5/2015
      Re-Gibson Assembly of Lac1 & Lac2 Jill, Suraj 8/6/2015
      PCR of Tyr Jill, Suraj 8/6/2015
      Gel of (Lane1: Ladder, Lane 2: Lac Gibson, Lane 3: Lac Gibson, Lane 4: Tyr1, Lane5: Tyr2, Lane6: Tyr3, empty, empty) Jill, Suraj 8/6/2015
      Gel of the rest of Tyr (140uL) (Lane 1: ladder, Lane2: Tyr, empty) Jill, Suraj 8/6/2015
      Gel Extraction of Lac Gibson (3.5ng/uL) Jill, Suraj 8/7/2015
      Re-measurement of Lac and Tyr concentrations using Cubit instead of nanometer Jill, Suraj, Melissa 8/26/2015
      PCR of Gibson Tyr and Lac Jill, Suraj 8/27/2015
      PCR of Tyr1, Tyr2, Lac1, Lac2 Jill, Suraj 8/27/2015
      Gel 1: Lane 1: Ladder, Lane 2: Lac1 PCR, Lane 3: Lac2 PCR, Lane 4 Tyr1 PCR, Lane 5 Tyr2 PCR -----Empty Melissa, Jill 8/28/2015
      Gel 2: Lane 1: Ladder, Lane 2: Lac Gibson A, Lane 3: Lac Gibson B, Lane 4 Tyr Gibson A, Lane 5 Tyr Gibson B ---- Empty Melissa, Jill 8/28/2015