Team:INSA Toulouse/contenu/lab practice/notebook/protocols/digest

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Notebook

Protocols

Digest Protocols

  • Our protocol
DNA Enzymes RNAse 10X Buffer H2O
5µL 0.5µL of each (1µL in total) 0.1µL 1µL (10% of total volume) 2µL (adjust at 10µL)

Do not overreach 10 % Volume with enzymes.

  • iGEM Restriction Digest Protocol

estimated time: 30 min. active, 50 min. incubation
The following protocol assumes you'll be doing restriction digests for 3A assembly, therefore we refer to your digests as:
· Part A (The 1st part in the future composite part)
· Part B (The 2nd part in the future composite part)
· Linearized plasmid backbone (The destination plasmid backbone for your composite part)
If you are simply doing a restriction digest for quality control, you can use the protocol below.
Restriction Digest from iGEM Videos.

Materials
· Ice and bucket/container
· (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
· Part A (Purified DNA, > 16ng/µl)
· Part B (Purified DNA, > 16ng/µl)
· Linearized plasmid backbone (25ng/µl)
· dH2O
· NEB Buffer 2
· BSA
· Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DpnI
· Thermal cycler

Notes
· You should keep all materials on ice.
· At iGEM HQ we use restriction enzymes from New England Biolabs

Procedure

  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 250ng of DNA to the appropriately labelled tube. Add distilled water to the tubes for a total volume of 16µl in each tube. 
Calculation example (with 25ng/µl as DNA sample concentration):
250ng ÷ 25ng/µl = 10µl of DNA sample
16µl (total volume) – 10µl (DNA sample) = 6µl of distilled water
  1. Pipet 2.5µl of NEB Buffer 2 to each tube.
  2. Pipet 0.5µl of BSA to each tube.
  3. In the Part A tube: Add 0.5µl of EcoRI, and 0.5µl of SpeI.
  4. In the Part B tube: Add 0.5µl of XbaI, and 0.5µl of PstI.
  5. In the pSB1C3 tube: Add 0.5µl of EcoRI, 0.5µl of PstI, and 0.5µl of Dpn1.
  6. The total volume in each tube should be approximately 20µl. Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture to the bottom of the tube.
  7. Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes. We use a thermal cycler with a heated lid.
  8. (Optional, but recommended) Run a portion of the digest on a gel (8µl, 100ng), to check that both plasmid backbone and part length are accurate.
  9. Use ~2µl of the digest (25ng of DNA) for ligations.
Part A Part B Linearized plasmid backbone
DNA 250ng 250ng 250ng (10µl @ 25ng/µl)
dH2O adjust to 16µl adjust to 16µl 6µl
NEB Buffer 2 2.5µl 2.5µl 2.5µl
BSA 0.5µl 0.5µl 0.5µl
Enzyme 1 0.5µl EcoRI 0.5µl XbaI 0.5µl EcoRI
Enzyme 2 0.5µl SpeI 0.5µl PstI 0.5µl PstI
Enzyme 3 0.5µl DpnI

Single Reaction

  1. Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16µl.
  2. Add 2.5µl of NEBuffer 2.
  3. Add 0.5µl of BSA.
  4. Add 0.5µl of EcoRI.
  5. Add 0.5µl of PstI.
  6. There should be a total volume of 20µl. Mix well and spin down briefly.
  7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid
  8. Run a portion of the digest on a gel (8µl, 100ng), to check that both plasmid backbone and part length are accurate.

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