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Project Documentation & Protocols: Maize Gene Discovery Project: RescueMu Library Plates: Protocols: Picking

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Plasmid Rescue

Genomic Sequencing Utilizing a RescueMu Insert:
Genomic DNA Extraction From a Transposed RescueMu Population

General Description: A plasmid rescue procedure for recovering restricted maize genomic DNA fragments containing RescueMu element insertion in plasmid form into E. coli cells.

 

Materials & Reagents:
DNA
10X BRL React buffer
RNase T1
BglII
BamHI
Phenol:Chloroform:Isoamyl alcohol (25:24:1)
Chloroform:Isoamyl alcohol (24:1)
3M sodium acetate
Ethanol - 95% purified
Laminar flow hood
ddH2O (deionized and distilled water)
5xBRL ligation buffer
T4 DNA ligase
DH10B ElectroMax (BRL #18290-015) cells
0.1 cm cuvette (BioRad)
SOC medium
LB +100 mg/L Carbenicillin plates

 

Procedure:

Restriction Digestion

Components
10 microgram DNA
20 microliter 10X BRL React 3
0.2 microliter RNase T1
50 units BglII
50 units BamHI
Final volume 200 microliter
Digest for 90 minutes at 37 C

Directions

  1. Add equal volumes of Phenol:Chloroform:Isoamyl alcohol (25:24:1) and mix by inverting the tubes for 1-2 minutes. Spin in a microcentrifuge for 10 minutes at maximum speed and carefully transfer the upper aqueous phase to a fresh tube.
  2. Repeat step 1.
  3. Repeat step 1 with Chloroform:Isoamyl alcohol (24:1) once.
  4. Add 20 microliter 3M sodium acetate and mix, then add 500 microliter ethanol and mix. Incubate on ice for at least 5 minutes and centrifuge for 20 minutes at maximum speed. Pour off ethanol carefully. Wash the pellet with 500 microliter 70% ethanol. Centrifuge for 5 minutes at maximum speed. Pour off ethanol carefully. Spin again briefly before aspirating the remainder of the ethanol. Dry the pellet in a laminar flow hood for 10-15 minutes.
  5. Resuspend the pellet in 300 microliter of ddH2O.

Ligation

Components
10 micrograms digested DNA
100 microliters fresh 5xBRL ligation buffer
10 units T4 DNA ligase
Final volume 500 microliters
Ligate at room temperature for 1 hour

Directions
  1. Purify DNA by repeating steps 1 through 4 for Restriction Digestion, above.
  2. Resuspend the pellet in 10 microliters ddH2O.

Electroporation

Components
2 microliter ligated DNA for each 30 microliter DH10B ElectroMax (BRL #18290-015) cells
0.1 cm cuvette (BioRad)
100 ohms, 2.3-2.5 kV, 25 microF (electroporation conditions)

Directions
  1. Immediately add 1 ml SOC medium to the electroporated cells.
  2. Grow (200 - 250 rpm) at 37 C for 45-60 minutes and then plate aliquots onto LB +100 mg/L Carbenicillin plates.
  3. Note: Colonies should be plated so that they are adequately separated from each other.


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