Team:Oxford/Protocols

Protocols

1.0 PCR (Polymerase Chain Reaction)

PCR is used to amplify a specific region of DNA.

Before starting:

  • Defrost DNA templates and primers
  • Use the 150\(\mu\)l aliquots of the Q5 Master Mix that are stored in the iGEM box in the 4℃ cold room. This avoids repeat freeze-thaw of the stock solution. Bring ice bucket to the cold room to bring Q5 to the bench.
  • Label PCR tubes

Reaction Mix:

Component Volume/\(\mu\)l Final conc/nM
Q5 HF Master Mix 12.5 -
10\(\mu\)M Forward Primer 1.25 500
10\(\mu\)M Reverse Primer 1.25 500
1ng/\(\mu\)l-1 1.0 -
Milli-Q Water 9.0> -

Reaction protocol:

This is an example, times may vary based on the polymerase used.

Stage Number of Cycles Temperature/℃ Time/min
Initial denaturation 1 98 2
Denaturation - 98 0.5
Annealing 25 Annealing temp. 0.5
Extension - 72 0.5 per 1kb
Final extension 1 72 5

During and after preparing tubes:

  • Make sure that the primer and small amounts of DNA do not stick onto the side of the tube or tip
  • Use the calculated annealing temperature

You are always going to want to run the PCR products on a gel. Set up the gel once you have started the PCR.

When the PCR has finished:

Add blue loading dye to each PCR tube (be careful not to add the purple ladder accidentally) according to the volume of PCR product you are running and type of stain. For example, if you are using the 5x dye and you're running 20\(\mu\)l PCR prodct, add around 4\(\mu\)l dte. '5x' refers to the total DNA solution volume compared to the loading dye.

Reaction mix for Phusion:

Component Volume/\(\mu\)l
Phusion Buffer 10
Phusion enzyme 0.5
dNTPs 5
Template 0.5
Milli-Q 34

1.1 Gel Electrophoresis

Fragment Size Agarose gel w/v % Mass of agarose in 200ml 0.5x TBE/g
>3kb 0.8 1.6
<1kb 2 4
In between 1 2

For a large 1% gel, prepare 200mL agarose

  1. Heat 2g agarose in 200ml 0.5x TBE for 2 minutes under full power in the microwave (use a 500mL Duran bottle, and place a weighing boat underneath it to prevent the causing of a mess in the event the mixture boils over; DO NOT fully tighten the Duran cap).
  2. Check if the agarose has been fully dissolved. Heat it further if gel strands are visible.
  3. Hold the lid with paper and gentlyswirl
  4. Leave the agarose solution to cool at 50℃ for 20 minutes.
  5. Pour agarose onto gel plate in a setting tray with appropriately-sized combs already fixed onto it, and leave for 20 minutes to let it set.
  6. When the agarose has set, remove the combs and transfer the gel plate from the setting tray to the electrophoresis chamber.
  7. Flood the gel plate with 0.5x TBE buffer up until right above the top of the wells.
  8. The gel should be positioned such that the positive (red) electrode is on the far side of the gel from the wells, as the negatively-charged DNA will migrate towards the positive electrode.
  9. Load 10\(\mu\)L DNA ladder in lane 1 and 20\(\mu\)L PCR product in subsequent lanes
  10. 120V for a big gel (200mL agarose) or 80V for a small gel(100mL agarose)

1.11 Staining the Gel

  1. Pick up the gel keeping it flat and allow the excess buffer to run off
  2. Using your hands, slide gel carefully into a vat of ethidium bromide
  3. Set the vat to gentle shaking for 30/40 minutes
  4. Pick up the gel using a spatula and rinse off the ethidium bromide

1.12 Visualizing DNA using UV Transilluminator

  1. Place the gel on the transilluminator stage and adjust stage height appropriately.
  2. Set the transilluminator using the GeneSnap program such that the light emitted is UV (instead of white light) and the software filter is configured to pick up EtBr fluorescence.
  3. Adjust the contrast such that the bands can be clearly seen.
  4. Adjust the focus using the focusing rings to sharpen the image.
  5. Save the image in the naming format “dd_mm_yy” to Disk C: → Lab users → iGEM in .sdg file format, and additionally export it as a .tif file.
  6. Print a picture off for your own records
  7. Label eppendorfs according to successful bands
  8. Excise bands and slide into appropriate eppendorf

1.13 Extraction of DNA (PCR product) from Gel

Remember, when spinning tubes with their lids open, place them so that lids are pointed away from the direction of spinning.

  1. Zero the weighing scale to weight of eppendorf
  2. Weigh each of the bands
  3. Dissolve excised chunks in a minimum of 1mL of XP2 Binding Buffer per gram of gel

Green box on our shelf - E.Z.N.A Gel Extraction Kit made by Omega Bioteck, according to the Spin Protocol.

Elute PCR products into 30\(\mu\)l and plasmid DNA into 50\(mu\)l.

1.2 Restriction Digest PCR or Plasmid DNA

  • Use enzymes and buffer according to "Master Table"
  • Defrost and shake buffers
  • Keep enzymes in yellow freezing block and keep out of freezer for as short a time as possible
Component Volume/\(\mu\)l
DNA 30
Buffer 5
EcoRI-HF 0.5
SpeI 0.5
Milli-Q Water 14

A point to note concerning the volume of restriction enzyme

  • 0.5\(\mu\)l for PCR DNA
  • 1.0\(\mu\)l for Plasmid DNa
  • However, if you are doing a test digest (i.e. after a mini-prep) use 0.5\(\mu\)l enzyme, despite digesting a plasmid
  1. Incubate at 37℃ for 2 hours (ThermoMixer program 3) with shaking at 300rpm.
  2. Heat inactivated for 30 minutes at 95℃
  3. Dephosphorylate the plasmid using 1\(\mu\)l CIP at 37℃ for 3 minutes

1.21 DNA 'Clean Up' using EZNA Enzymatic Reaction Kit

Protocol can be found at the end of ENZA gel extraction booklet.

Elute PCR products into 30\(\mu\)l and plasmid DNA in 50\(\mu\)l.

1.22 Nanodrop

  1. Clean stage with 1\(\mu\)l water and tissue
  2. Make a blank reading using 1\(\mu\)l of water and wipe off
  3. Make another blank reading using 1\(\mu\)l of elution buffer and wipe off
  4. Measure concentration of 1\(\mu\)l of each sample

1.3 Ligation

Overnight protocol

Defrost T4 DNA Ligase on ice.

Keep in freezing block when on bench and add last to the reaction mixture.

Mass of vector DNA : Mass of insert DNA roughly 1:3

Generally, because you only get 50\(\mu\)L from the plasmid digest, split evenly between how many inserts there are.

The component volumes are:

Component Volume\(\mu\)l
Digested DNA (gBlock) 29
Digested pSB-1C3 7
T4 DNA Ligase Buffer 5
T4 DNA Ligase 1
Milli-Q 8

Incubate at 16℃ overnight.

1.4 Preparation of Competent E.coli Cells

Overnight protocol

DH5ɑ is stored at -80℃

  1. Defrost and inoculate in 5mL of LB in a 125 mL conical flask (volume of LB 10% of flask volume so as to achieve sufficient aeration)
  2. Grow culture overnight at 37℃

Next Day

Turn on the centrifuge and cool to 4℃

Always keep TFBI and TFBII on ice.

Important to keep volumes accurate; else your cells will grow at different rates and ODs will be all over the place.

  1. Transfer 1ml of overnight culture into 100ml LB in a 500ml conical flask.
  2. Incubate at 37℃ until OD600nm reaches 0.4-0.6. It is good to stop at OD = 0.35, as the bacteria are now replicating exponentially i.e. will only take 20 more minutes until OD = 0.7 (which is far too high).
  3. While incubating, pre-chill 20 eppendorfs and 2x50mL Falcon tubes (which are found in the cupboard next to the sink) to 4℃ in ice bucket.
  4. Once OD is correct, decant culture into 2 x 50ml falcon tubes. Once this is done, the cells must never be higher than 4℃.
  5. Centrifuge at 2000 rpm at 4℃ for 20 mins. Make sure you are at the centrifuge when this 20 minutes is up to rapidly proceed to the next step. Close the centrifuge lid to maintain 4℃.
  6. Discard supernatant and resuspend pellets in 1ml TFBI.
  7. Add further 10ml TFBI using 10ml electronic pipette.
  8. Leave on ice for 20 mins (or more).
  9. Centrifuge at 2000 rpm at 4℃ for 10 mins (shorter duration as cells are already now permeabilized).
  10. Discard supernatant.
  11. Gently resuspend pellet in 2ml TFBII i.e. don’t vortex, just flick pellets in the TFBII, which has glycerol for frost protection (prevent crystal formation which lyses cells).
  12. Now we have total 4mL of cell-TFBII suspension. Aliquot ~200ul into ~20 pre-cooled eppendorf tubes.
  13. Store at -80℃ in the freezer in the back room.

1.5 Transformation

Overnight Protocol

To transform cells you need to prepare agar plates. This can be done when you have a spare hour during the day. Plates can be left of the bench (lid side down) until you need them. Plates can also be made during the transformation protocol.

200ul aliquots of competent cells are stored at -80C in the back. These take around 30 minutes to defrost and so these need to be taken out of the freezer and put on ice to defrost. 100ul of competent cell = 1 transformation.

Preparing the cells for transformation

For ligated DNA:

  1. Put the eppendorfs of ligated DNA into ice to cool to 4℃.
  2. Draw 100\(\mu\)l of competent cells of each aliquot to each 50ul ligation product.
  3. Incubate on ice for 30 minutes. If you are preparing plates as you go along, the LB agar should be done microwaving at this point.
  4. Bring ice bucket to the water bath.
  5. Heat shock - transfer straight from ice bucket into 45℃ water bath for 45 seconds.
  6. Transfer back to ice for 1 minute.
  7. Add 800\(\mu\)l LB broth to each tube and incubate at 37℃ gently shaking for 1 hour.

For plasmid DNA:

  1. Thaw the plasmid DNA for transformation or use ligation product.
  2. Pre-chill number of eppendorfs to match number of transformations.
  3. Thaw appropriate number of 200\(\mu\)l aliquots of competent E. coli cells to prepare 100\(\mu\)l eppendorfs to match number of transformations.
  4. Draw 100\(\mu\)l out of each aliquot (competent cells) and transfer into empty pre-chilled eppendorfs.
  5. Transfer the full volume (usually ~50\(\mu\)l) of the plasmid DNA into the respective E. coli aliquots (we use all of it as we expect the plasmid concentration to be relatively low). For the stock pSB1C3, add only 1\(\mu\)L of the plasmid to the 100\(\mu\)L of cell suspension as the plasmid is at much higher concentration than for e.g. ligation products.
  6. Incubate on ice for 30 minutes. If you are preparing plates as you go along, the LB agar should be done microwaving at this point.
  7. Bring ice bucket to the water bath.
  8. Heat shock - transfer straight from ice bucket into 45℃ water bath for 45 seconds.
  9. Transfer back to ice for 1 minute.
  10. Add 800\(\mu\)l LB broth to each tube and incubate at 37℃ gently shaking for 1 hour.

1.51 Preparing the Plates

In the laminar flow hood
  1. LB is in agar form on the shelf
  2. Label the bottle
  3. Loosen the lid, place on plastic dish and microwave on simmer for 20 mins. Melt LB agar as cells are being thawed. Each 500mL bottle of agar makes ~20 plates
  4. Prepare 30 mg/ml chloramphenicol in EtOH e.g. 300 mg chloramphenicol in 10ml EtOH and 100 mg/ml ampicillin solution in MilliQ e.g. 1g ampicillin in 10ml MilliQ
  5. Cool in 50℃ water bath 30 mins for smaller bottle, slightly longer for larger ones. The bottle is cool enough when you can just about comfortably carry it to the laminar flow hood

Add x mL media to x uL antibiotic e.g. 100ul chloramphenicol solution to 100ml agar. Poor the plates accordingly. Place close to the wall of the hood to prevent contamination. Agar takes around 20 mins to solidify.

1.52 Spreading Plates

How you do this depends a bit on how many plates you are spreading but the idea is to pipette cells onto the plate and spread using glass beads, without leaving too much time between these two steps so that the cells dry in a drop in the middle.

Sterile Technique
  1. Pipette 100\(\mu\)l of the cell onto the plate
  2. Flame the mouth of the bottle that contains the glass beads and tip around six onto the plate, flaming the mouth again before putting the lid back on (don’t put the lid down on the bench)
  3. Move the plate until streaks from glass beads fill the plate
  4. Discard the glass beads using the funnel into alcohol (located at the end of the bench near the heating blocks)
  5. Spin down the remaining E. coli (max speed, 1 min) and repeat steps 1-4 onto a separate plate (label so as to distinguish between plates clearly)
  6. Incubate upside down at 37℃ overnight, clearly labelled with date

1.6 Growth and Culture of Bacteria

Overnight protocol

This process significantly increases the amount of plasmid that contains biobrick that we want. Plasmids can be extracted later.

Antibiotics are in the freezer - ampicillin needs defrosting before you start.

The LB you use has to be transparent (cloudy = contaminated).

Sterile Technique

The tip of each test tube and Duran bottle must be sterilised with bunsen burner each time when you are preparing the tubes and each time you transfer a colony.

[Arrange your hands in a way that you have your thumbs free and lids do not get placed on the desk. This is difficult to explain so ask someone who has done it before to show you.]

  1. Choose three colonies from each plate. The colony should not be too small or too large and should be reasonably spaced from the others.
  2. Label a test tube for each colony.
  3. Pour 5ml LB broth that has antibiotic to be diluted 1/1000 fold to each tube i.e. if you have 10 plates and you are preparing 30 tubes, you will need 30x5 = 150ml LB with 150\(\mu\)l antibiotic already added. Therefore, each tube contains 5ml LB and 5ul antibiotic
  4. Using inoculation spatulas, pick colony and transfer to appropriate tube. When taking an inoculation spatula from the packet, be careful not to reach into the packet; instead, push handle out of packet to keep sterile
  5. Push down and bring the colony directly into LB without touching the sides
  6. Remove the test tubes from the rack and place in 37℃ overnight

1.7 Mini-prep

E.Z.N.A. Plasmid DNA Mini Kit I

If you haven’t done a mini-prep before ask someone who has to go through it with you

  • Carry out all optional steps except equilibration step
  • Repeat 1st centrifugation (step 2) until all the LB broth has been spun down and all the E. colihave been collected - will help increase yield later
  • After centrifugation in step 2, pulse tubes before the excess supernatant was removed through pipetting
  • After addition of solution I/RNAse, vortexing/vigorous shaking of the tubes should be avoided to prevent shearing of nucleus and undesirable accidental extraction of chromosomal DNA.
  • After addition of solution I/RNAse, resuspension of pellet can be done by dragging the tube along an eppendorf rack.
  • Steps 6 and 7 (involving solutions II and III need to be carried out in quick succession (adhering to the short incubation time) to ensure good results. It is advisable to do these two steps in pairs as in step 6 the tubes need to be tightly capped once solution II is added
  • The inversion in step 6 needs to be done gently so that genomic DNA of the bacteria are not extracted along with the desired plasmid DNA
  • After addition of solution II, the waiting time before proceeding to the next step should not be more than 5 minutes
  • The inversion in step 6 needs to be done gently so that genomic DNA of the bacteria are not extracted along with the desired plasmid DNA
  • The precipitate formed in following step 7 does not pellet well after centrifugation in step 8, and hence the supernatant needs to be removed immediately to prevent resuspension

  • Elution step:

    • place elution buffer in 55℃ water bath (50\(\mu\)l per miniprep)
    • pipette warmed elution buffer onto the HiBind column and let it sit for 3 mins
    • centrifuge at max speed for 1 minute
    • pipette up filtrate and pipette back onto HiBind column
    • centrifuge at max speed again

→ Nanodrop → Restriction Digest → Gel electrophoresis → Sequence

2.0 TCA Protein Precipitation

Stock Solutions

100% (w/v) Trichloroacetic acid (TCA)

recipe: dissolve 500g TCA (as shipped) into 350 ml dH2O, store at RT

Precipitation protocol

  1. Have bacteria grown in appropriate antibiotic-supplemented media to stationary phase the night before.
  2. Dilute an aliquot of the stationary phase bacteria (1/20 dilution) in an appropriate antibiotic-supplemented media and grow to desired OD600 (typically: ~0.6 - 0.8; for our E. coli it takes about 1 - 1.5 hours in LB at 37℃).
  3. Add an appropriate amount of arabinose to achieve a final concentration of 0.2% (e.g. 200\(\mu\)l to 20ml) in the culture and incubate further (1 hour gives some secretion, 4 hours should give extensive secretion).
  4. Spin down 1.5mL of the cell cultures at full-speed for 5 minutes, and transfer 1.35mL of the supernatant into a separate microcentrifuge tube.
  5. Add 150\(\mu\)L of 100% TCA into the supernatant, vortex mix, and centrifuge it at full speed for 15 minutes in the cold room. Put acetone in ice in the meantime.
  6. (Stay in the cold room) After centrifugation is complete, discard the supernatant and add 900\(\mu\)L of ice-cold acetone to each tube. Vortex/shake briefly to wash off remaining TCA from the pellet, and spin down again for 5 to 10 minutes at full speed.
  7. Discard supernatant and let pellet dry for about 30 minutes/place in 95℃ heat block to drive off acetone

2.1 SDS-PAGE

Running the gel

  1. Prepare 1x SDS loading dye containing a final NaOH concentration of 50\(\mu\)M.
  2. Resuspend the pellets using 30\(\mu\)L of said dye, and boil at 99℃ for 5 minutes or until the pellet dissolves to give a blue solution.
  3. If the resulting solution is yellow, adding 5\(\mu\)L of 20 mM NaOH should turn it blue. Be careful when opening lids of hot eppendorfs - if the interior of the tube is steamy, let it cool down and subsequently spin it down to pull down the liquid first.
  4. Obtain PAGE gel cassette from cold room, and dilute some 20x SDS buffer (found on top of bench) to 1x (800mL typically needed to fill PAGE tank). NB: Use the tank without the yellow strips on the inside.
  5. PAGE tank can be found in one of the cabinets at Tom’s bench.
  6. Load cassettes and 1x buffer into tank.
  7. Obtain ladder (SDS 2-colour dye) from Jia’s freezer, load 15uL into well in cassette.
  8. Load 15\(\mu\)L of each sample into wells.
  9. Run gel for at least 40 minutes at 100mA per gel.
  10. Once the dyefront is at a desirable position, remove gel from cassette and place it in a square petri dish. Stain with Instant Blue for 1 hour but preferably overnight.