Arkin:JCAProtocols Shuffling

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DNA Shuffling

This is the DNA Shuffling (or Sexual PCR) described by Stemmer. The DNA fragments are sheared into ~50bp fragments, then reassembled into a full-length library giving all permutations of combined mutations present in the original sample. It also induces additional point mutations and is therefore also a good error-prone pcr method.

DNase I Solution

 2.0 Units/ml DNase I
 50 mM Tris-HCl, pH 7.4
 Prepare just before use
 10 mM MnCl2

Note: The Qiagen RNase-Free DNase Set provides vials at 1500 units, dissolved 550 uL, so the concentration is 2.7 units/uL (2700 units/mL)


Fragmentation

  1. Dissolve 2 to 5 μg of parent DNA in 10 μl of ddH2O (see Hint #1).
  2. Incubate the solution at 15 °C.
  3. Prepare 40 μl of DNase I solution and incubate at 15°C.
  4. Add 10 μl of the parent DNA solution to the 40 μl of DNase I solution at 15°C.
  5. Transfer 10 μl aliquots from the digestion reaction to individual tubes on ice containing 1 μl of 0.5 M EDTA, urea, and dye after 1, 2, 3, 5, and 10 min of incubation. (see Hint #2).
  6. Electrophorese the samples on a 12% TBE-Urea PAGE minigel along side an oligo sample around 50bp
  7. Place the gel on a TLC plate covered in saran wrap, cut the region similar in size to the 50bp oligo, put it in a microcentrifuge tube
  8. Add 500 uL or so of 1xTAE, and let shake at 37 for at least 2 hours to overnight to leach out the DNA fragments into solution
  9. Filter the liquid through a miniprep column or 0.2 micron column to remove bits of gel
  10. Measure the volume with a pipettman. Add 1/10th volume of ethanol and 2.5 volumes of ethanol. Chill 10 minutes at -20.
  11. Pass the liquid through a Zymo column or Qiagne blue column to collect the DNA, wash 2x with Qiagen PE Buffer or Zymo Wash Buffer to remove salts, spin 1 minute to dry, elute in 10 uL water

Assembly

1. Combine the following in a microcentrifuge tube:

  5 μl of 10X Taq Buffer
  5 μl of dNTP Mix
  10 μl of the purified fragments
  2.5 Units of Taq Polymerase
  Add ddH2O to a total volume of 50 μl.

2. Incubate the reaction for 3 min at 94°C.

3. Amplify the DNA fragments in a thermocycler with the following program:

  30 sec at 94°C
  1 min at 55°C
  1 min at 72°C
  Lengthen each extension step by an additional 5 sec per cycle.
  Repeat for 40 cycles (see Hint #6).

4. Visualize a small portion of the reaction by electrophoresis on a 2% (w/v) Agarose Gel (see Protocol ID#1265 and Hint #7).

Amplification

1. Mix the following components in a microcentrifuge tube:

  10 μl of 10 X Taq Buffer
  10 μl of dNTP Mix
  0.5 μM each Primer (Primer #1 and Primer #2)
  1 μl of Assembly Reaction from Step #B3
  5 Units of Taq Polymerase
  Add ddH2O to a total volume of 100 μl.

2. Amplify the DNA fragments in a thermocycler with the following program:

  30 sec at 94°C
  30 sec at 55°C
  30 sec at 72°C
  Repeat for 30 cycles (see Hint #6).

3. Visualize a small portion of the reaction by electrophoresis on a 2% (w/v) Agarose Gel (see Protocol ID#1265 and Hint #8).

4. Electrophorese the DNA on a 2% (w/v) preparative Agarose gel (see Protocol ID #1265 and Hint #3) containing 0.5 μg/μl Ethidium Bromide (CAUTION! See Hint #9) to purify the DNA sequences for cloning into an expression vector.

Adapted from http://www.bio.com/protocolstools/protocol.jhtml?id=p2189

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