Team:Oxford/Test/Protocols

Protocols

1.0 PCR (Polymerase Chain Reaction)

PCR is used to ampilfy 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 tues

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
Inital 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 te primer and small amounts of DNA do not stick onto the side of the tube or tip
  • Use the calculated annelaing temperature

You are alwasy 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 dye. '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 accoring 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 libs 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 Restricion 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 inavtivate 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, beacuse 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.