Assembly Related Protocols

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Contents

Combining basic part with assemblies

  1. PCR
  2. Purfication using gel purification (cut out the gel band)OR do the "lazy way" + zymo.
  3. Digest
    • basic part
      • EcoRI/BamHI/DpnI
      • Digest with 50 ul scale
    • Assembly + vector
      • take 4ul part
      • 4ul water
      • 1 ul NEB2
      • 0.5 ul EcoRI
      • 0.5 ul BglII
  4. ZYMO BOTH simultaneously
      • part: elute with 10ul water
    • AVector: elute with 6ul water



Newest EcoRI/BamHI Transfer method (7/31/08)

Digestion:

  • 0.5ul plasmid in entry vector
  • 0.5ul EcoRI
  • 0.5ul BamHI
  • 1ul NEB2 + ATP buffer
  • 7.5ul of water
  • incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill.

No purification step.

Ligation:

  • 1ul of dilute assembly vector (or 0.5ul if undiluted)
  • 0.5ul ligase

Transform. (same as usual)

Transfers from Miniprepped DNA

(already in correct vectors - transfers into L/R cells)

In 0.5 ml tube, combine:

  • 1ul plasmid dna
  • 30ul of cells (L/R) + 30 KCM (no water)

Take the reaction, and:

  • sit in ice for 10 min
  • heat shock for 90 sec
  • sit another 2 min ice

Then, incubate your tubes for 5 minutes (No rescuing is necessary)

Next, prepare plates (double antibiotics) and tubes with media (LB + antibiotics)

  • Take 15ul of rxn. Spot it onto a plate (no spreading required)
  • Take the remaining 15ul into a pipette tip. Drop the entire tip into your tube of media.
  • Let plates and tubes sit overnight.
  • Miniprep the next day.


1-2-3 Assembly

Set up the digestion:

  • 1uL lefty plasmid(methylated)
  • 1uL righty plasmid (methylated)
  • 1uL NEB2+ATP
  • 0.3 uL XhoI
  • 0.3 uL BglII
  • 0.3 uL BamHI
  • 6.1uL Water

Heat kill at 65 for 20 min

Add 0.3uL Ligase

Transform, rescue, plate on dual antibiotic plates

Dealing with Co-transformation (8/4/08)

Method 1: Pick more white colonies...

Method 2: Seperating the 2 plasmids:

  1. Take miniprepped, cotransformed dna sample, and dilute 100x
  2. Take 1ul of diluted DNA to transform
    • Use selected cell strain
    • rescue 15 min in incubator
    • Grow on double-antibiotic plate overnight
  3. pick colonies (maybe 2)
  4. streak on double antibiotic plate, and amp or spec to check for co-transformation
  5. simultaneously grow in media with corresponding double antibiotics
  6. miniprep the next day

Method 3: "Removing" the cotransformant plasmid

  1. pick colonies from the restreaked plate (the ones that are for sure cotransformed
  2. grow in double antibiotic media overnight
  3. dip toothpick into culture from night before, and restreak on double antibiotic plate
  4. the next day, pick colonies and screen for cotransformation



Sherine Cheung
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