Team:LUBBOCK TTU/Experiments

  Protocols (Experiments)

This page is for the protocols we used over the course of this project. Below we have split into categories Medias, PCR, Cloning, Digestion/Ligation, and Kit Protocols. In each category we have several protocols that can be used in conjunction or separately of each other. We hope that these protocols would provide useful to others that try to replicate our projects, parts of our project, or find a certain protocol helpful in their own experiment.

· dH2O 100mL
· Tryptone 10g
· Yeast extract 5g
· NacL 10g
· Agar 10g
· *1 mL of 50mg/mL ampicillin for 50ul/mL ampicillin LB agar plates
· *1mL mL of 50mg/mL chloramphenicol for 50ul/mL chloramphenicol LB agar plates

1. Boil on hot plate and stir with magnetic stirrer to fully dissolve
2. Autoclave for at least 15 mins at 121C
3. Wait for it to cool (but not solidify) before pouring into plates (~18-20mL in each plate)
4. If using ampicillin or chloramphenicol, add it to the cool agar and mix right before pouring the plates
5. *any extra can be discarded into the biohazard trash once it has solidified or left in the bottle and warmed up again to pour again, just make sure bottle lid is closed shut

· dH2O: 1000mL
· YPD broth mix: 50g
· Yeast extract: 10g
· Peptone: 20g
· Dextrose: 20g
· Agar: 15g

1. Boil on hot plate and stir with magnetic stirrer to fully dissolve
2. Autoclave for at least 15 mins at 121C
3. Wait for it to cool (but not solidify) before pouring into plates (~ 18-20mL) in each plate

· dH2O: 1000mL
· YPD broth mix: 50g
· Yeast extract: 10g
· Peptone: 20g
· Dextrose: 20g

1. Mix fully until dissolved
2. Autoclave for at least 15 mins at 121C
3. Wait for it to cool before using it

· dH2O: 900mL
· 8g selective media
· 20g bactoagar

1. Stir until dissolved
2. Autoclave at 121C at 15 mins
3. Add approximately 100mL glucose when cool

· Q5 High Fidelity Master Mix
· Forward/Reverse Primer (100mm)
· Dilute to 10umm for PCR
· Nuclease Free Water
· DNA to be run in PCR

1. Create “master mix” for appropriate amount of samples

2. Dispense appropriate amount in each PCR tube
3. Add appropriate amount of DNA to each tube

Below is the PCR template we used for our fragments/custom genes
For this template, each PCR tube contained the following:
· 7.5ul nuclease free water
· 12.5ul Q5 high fidelity master mix
· 0.25ul forward primer
· 1.25ul reverse primer

4. Result in Total volume of 22.5ul before DNA is added
5. 2.5ul of DNA is added for Total volume of 25ul
6. Thermocycler programed according to several PCR test runs to find ideal annealing temperature

For all our fragments, ideal temperature was 60C (as denoted in red on the template)
For some of our fragments, the extension time was extended according to the amount of kb in the gene (30 minutes per 1 kb according to the user protocol for the Q5 master mix)

Items needed:
· TAE X1 or TBE X1
· Agar
· Gel casting tray
· Gel electrophoresis apparatus set up
· Comb for wells
· Sybr Safe or Ethidium Bromide

1. Make mix for gel
- a. Mix .8g of agar and 80mL of TAE X1 in beaker
- b. Double boil using microwave, swirling periodically
2. Once removed from microwave, wait for it too cool before adding 2ul of Ethidium Bromide
3. Once it has cooled, pour into the gel cast mold, using a comb with enough wells for your sample plus two wells for ladders
4. Wait 15-20 minutes for gel to solidify
5. Once the gel has solidified, remove from the caster and place into the electrophoresis box properly orientated with the negative side being closer to the wells
6. Fill the box with TAE X1 until the wells are completely covered
7. If not purifying afterwards, mix 2ul of loading dye into each sample to be run in the gel
- a. If purifying afterwards then aliquot out 5ul of sample in a separate tube to mix with the loading dye
8. Aliquot out 10ul of ladder in separate tube and mix with 4ul loading dye
9. Starting with ladder, load the wells with 7ul of sample, ending with the second ladder
10. Connect the gel box to a voltage and run for approximately 45 minutes at 130-150V
11. Once the bands have migrated about 3/4 down the gel, turn off the power source and image the gel with a UV imager

1. Take the complement of the beginning of the strand you're trying to synthesize, this will be your forward primer
- a. Ideal length of primer is 16-30 bases
2. Take the reverse complement of the end of the strand you're trying to synthesize, this will be your reverse primer
- a. Ideal length of primer is 16-30 bases
3. Use an online CG calculator to find the melting temperature of your primer (ideally between 60-65C)
*Make sure your primers are within the same range of Melting Temperatures so that you can set the proper annealing temperature

1. Use IDT’s calculator to check for hairpin loops
2. If everything checks out then order using IDT, cost is usually under $20

The following is an abbreviated guide for the Thermo Fisher TOPO cloning Kit

Items Provided in Kit: · Salt Solution
· Water
· TOPO vector

Items not provided:
· PCR product
· Taq Polymerase to re-Taq if necessary
· Top10 competent E.Coli cells

1. In a tube mix 1ul of Salt Solution, 1ul of TOPO vector, 0.5-4ul of PCR product, and enough water so the solution equals a total of 6ul
2. Mix the reaction and incubate at room temperature for 5 minutes
3. Afterwards, transform into competent cells using the Top10 Cell transformation protocol
4. Plate the transformed cells onto a LB ampicillin or a LB kanamycin plate

*Your PCR product must contain taq polymerase for this protocol
*The cloning must be done within 20 minutes of the PCR finishing or else the product must be re-taqed
*To re-taq, add 0.7-1 units of taq to the PCR product and incubate in a heat block at 72C for 8-10 minutes

Items needed:
· Competent Top10 E. coli
· Water bath
· DNA to be transformed
· LB broth or SOC media
· LB Ampicillin or LB Kanamycin plates

1. Turn on water bath to 42C
2. Add 100ul competent cells to tube
3. Add 1-10ul of DNA to be transformed to the same tube
4. Let the tube sit in ice for 15-20 minutes
5. Heat shock for 30 seconds at 42C
6. Place back in the ice for 1 minute
7. Add 250ul of LB media or SOC media to recover cells
8. Let sit in 37C incubator for 1 hour
9. Transfer the cells on an ampicillin or kanamycin (depending on resistance) and grow in incubator at 37C for 1-3 days

The following protocol was used to clone our fragmented gene together because it was too big to be synthesized by IDT

1. Use 10-100ng of each DNA fragment according to the guidelines in the protocl
- a. We used 10-25ng due to our fragments being 1-5kb
2. Thaw the provided Gibson Assembly Master mix on ice
3. In a PCR tube, prepare DNA fragments to the guideline with nuclease free water
4. Vortex before use when it has thawed
5. In a tube on ice, combine 5ul of DNA fragments and 5ul of Master Mix for total volume of 10ul
6. Mix by pipetting up and down
7. Incubate at 50C for an hour (we did this in a water bath)
8. After incubation, store at –20C until ready to use.

Items needed:
· 5 ul NEB Buffer 2 (We used NEB Buffer 4)
· 0.5 ul BSA
· 0.5 ul EcoRI-HF
· 0.5 ul PstI
· 0.5 ul DpnI (Used to digest any template DNA from production)
· 18 ul dH20
· pSB1C3

1. Create a Master Mix of everything but the linearized backbone
2. Add 4ul of the linearized backbone to a PCR tube
3. Add 4ul of the Enzyme Master Mix to the PCR tube
4. Digest at 37C/30 minutes
5. Heat kill at 80C/20 minutes

1. Take digested plasmid backbone 2ul and add equimolar of EcoRI-HF Pstl digested fragment (<3ul)
2. Add 1 ul of T4 DNA ligase buffer
3. Add 0.5 T4 DNA ligase
4. Add water to equal 10 ul
5. Ligate at 16C for 30 minutes
6. Heat kill at 80C for 20 minutes
7. Transform with 1-2 ul of product

Items needed:
· 50% PEG
· 1M LiAC
· Carrier DNA
· Sample DNA
· Sterile H2O
· Centrifuge
· Heatblock

Recipe for Master Mix:
· 50% PEG: 240ul
· 1M LiAC: 36ul
· Carrier DNA: 10ul
· Sample DNA: 2-4ul
· Sterile H2O: 74 ul

1. Grow cultures 50mL YPD overnight until they reach log phase
2. Place in falcon tubes, spin for 5 minutes @ 14K xg
3. Discard supernatant
4. Put 25mL sterile H2O and vortex
5. Spin again at 14K xg for 5 minutes
6. Discard supernatant
7. Resuspend pellet with 0.1M LiAC
8. Aliquot master mix into tubes, 300ul in each
9. Spin tubes at 15K xg to try to get rid of LiAC
10. Get rid of supernatant
11. Add 360ul master mix and resuspend pellet
12. Heat block for 30 mins at @ 30C
13. Remove and flick to mix
14. Incubate at 42C for 30 minutes
15. Centrifuge at max for no more than 25 seconds
16. Discard supernatant
17. Reuspend in 400ul dH2O gently
18. Plate 200ul on selection plates
19. Use glass beads to spread
20. Incubate at 30C for 2-4 days

The following is an abbreviated guide of the Sigma Aldrich mini prep kit used to isolate plasmid DNA from our cultures

Items provided by kit:
· Resuspension Solution
· RNase A Solution
· Lysis Solution
· Neutralization/Binding Solution
· Column Prep Solution
· Wash Solution
· Elution Solution
· Mini prep binding columns
· 2mL collection tubes

Items not provided:
· Ethanol (95–100%)
· Microcentrifuge
· Microcentrifuge tubes

1. Pellet 1-5mL of overnight culture by centrifugation
2. Discard the supernatant
3. Resuspend cells with 200ul Resuspension Solution, mixing by pipetting up and down
4. Lyse the resuspended cells by adding 200ul of the Lysis Solution
5. Mix the contents by gentle inversion until the mixture becomes clear and viscous (DO NOT VORTEX)
6. Precipitate cell debris by adding 3560ul of Neutralization/Binding Solution
7. Invert tube 4-6 times
8. Pellet Cell debris by centrifuging at 12,000xg for 10 minutes
9. Insert GeneElute Miniprep Binding Column to a microcentrifugation tube and add 500ul Column Prep solution
10. Centrifuge at 12,000xg and discard waste
11. Transfer lysate from step 3 to column prepped in previous steps and centrifuge at 12,000xg for 1 minute
12. Discard waste
13. Add 750ul of Diluted Wash to column
14. Centrifuge at 12,000xg for 1 minute
15. Discard waste and centrifuge again for additional minute
16. Transfer the column into a fresh collection tube
17. Add 100ul Elution Solution
18. Centrifuge at 12,000xg for 1 minute
19. Remove column and store DNA if not going to be used immediately at -20C

The following is an abbreviated guide for the Sigma Aldrich Midi prep Kit

Items included in the Kit:
· Column Prep Solution
· RNAse A Solution
· Resuspension Solution
· Lysis Solution
· Neutralization Solution
· Binding Solution
· Wash Solution 1
· Wash Solution 2
· Elution Solution
· Midiprep filter syringes
· Binding columns
· Collection Tubes

Item not included:
· Ethanol (95-100%)
· Centrifuge
· Vacuum Manifold (The vacuum method was the one used)

1. Place binding column into vacuum Manifold and apply vacuum
2. Add 4mL of Column Preparation Solution
3. Hold syringe barrel over binding column and gently apply pressure to the plunger to expel cleared lysate into column
4. Add 4mL of Wash Solution 1 to the column and allow it to pass through
5. Add 4mL of Wash Solution 2 to the column and allow it to pass through
6. Leave the column on the vacuum for 10 minutes to dry the column (if there are more than 6 columns then dry for 20 minutes)
7. Transfer the binding column to a collection tube and add 1mL of Elution Solution
8. Centrifuge in a swinging bucket rotor at 3,000xg for 5 minutes
9. If not using immediately then store at -20C

DNA purification was done by using the Sigma Aldrich PCR Cleanup Kit (70 Reactions)

The following is an abbreviated guideline of the protocol that comes in the kit and that was used

Items provided in kit:
· Column Prep solution
· Binding Solution
· Wash Solution
· Elution Solution
· Columns (in silver bag)
· 2mL tubes
· Items not provided in kit:
· 1.5mL Eppendorf tube
· 48mL 100% Pure Ethanol

1.Using the 2mL tubes provided in the kit, take a tube (1 per product to be purified) and place a provided column in the tube. Pipette 500ul Column Prep Solution into the tube and close
2.Centrifuge tube at 12,000xg for 1 minute
- a. The Column Prep solution should be at the bottom of the tube afterwards
3. After centrifuging, open the tube, pull out the column, discard in the Column Prep solution at the bottom at the tube into a waste container
4. Place the column back into the 2mL tube
5. In a separate 1.5mL tube, pipette 5ul of binding solution for every 1ul of product to be cleaned up
- a. Ex. 25ul of PCR product= 225ul of Binding Solution to be used
6. In the same tube that has the Binding Solution, add PCR product, and pipette up and down to mix
7. Take total volume from the tube that now contains the Binding Solution and PCR product and transfer to the 2mL collection tube with the column
8. Close the 2mL collection tube and centrifuge again at 12,000xg for 1 minute
9. After centrifuging, open the tube and pull out the column, discarding the solution that has accumulated at the bottom of the tube into a waste container
10. Place the column back into the tube and then pipetting 500mL of the Wash solution (After 48mL of the ethanol has been added) into the tube and then centrifuge for 1 minute at 12,000xg
11. Discard the waste from first wash and then centrifuge again at 12,000xg for 2 minutes to get rid of any remaining ethanol
12. Take out the column and then place it in a new 2mL, discard the old 2mL tube.
13. Pipetting 50ul of elution solution into the column and let it sit at room temp to incubate for 5 minutes
14. Centrifuge for 1 minute at 12,000xg
15. Afterwards discard the column and store at -20C if not going to use immediately