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Semi-Arbitrarily-Primed PCR to Identify Transposon Insertion Sites

Primers

Select one ARB primer and one transposon-specific primer for each round.

Transposon Round Primer Sequence (5'-3')
Any 1 ARB1 GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT
Any 1 ARB6 GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCC
Any 2 ARB2 GGCCACGCGTCGACTAGTAC
pMJM10, pEVS170, pEVS168 1 170Ext GCACTGAGAAGCCCTTAGAGCC
pMJM10, pEVS170, pEVS168 2 M13 For (-20) GTAAAACGACGGCCAGT
pMarVF1 1 MJM-440 TCAACACACTCTTAAGTTTGCTTC
pMarVF1 2 MJM-477 TTCCATAACTTCTTTTACGTTTCC
pJNW684 1 MJM-715 CATCCTGTCTCTTGATCAGAGCTT
pJNW684 2 MJM-716 CTTCCCAACCTTACCAGAGG

Use the transposon-specific Round 2 primer for DNA sequencing.

Primer notes

MJM-477 (pMarVF1): Sequence is in the direction opposite that of the erm cassette (i.e., plus direction sequence indicates that the erm cassette is inserted into the chromosome in the minus orientation).

MJM-716 (pJNW684): Sequence is in the direction opposite that of the kan cassette (i.e., plus direction sequence indicates that the kan cassette is inserted into the chromosome in the minus orientation).

DNA Preparation

Prepare genomic DNA (gDNA) from each candidate using the Qiagen DNeasy Blood & Tissue Handbook, using the Gram negative pretreatment. Elute in 100 μl H2O, determine DNA concentration by Nanodrop, and normalize the concentration to 28.6 ng/μl.

Semi-arbitrarily-primed PCR, Round 1

Recipe is for 50 μl reactions. Prepare master mix of a minimum of 10 reactions to avoid pipetting tiny volumes.

Arb-PCR, Round 1 50 μl Reaction Master (n=20)
5X GoTaq Reaction Buffer (colorless) 10.00 200
dNTP Mix, 2.5 mM each 4.00 80
primer1 (50 μM) - ARB1 0.50 10
primer2 (50 μM) - Tn-specific1 0.50 10
GoTaq DNA Polymerase (5u/μl) 0.25 5
H2O 27.75 555
gDNA of candidate (28.6 ng/μl) 7.00 --

ARB1

- 1 cycle:
    - 95 °C, 5 m
- 5 cycles:
    - 94 °C, 30 s
    - 30 °C, 30 s
    - 72 °C, 1 m 30 s
- 30 cycles:
    - 94 °C, 30 s
    - 45 °C, 30 s
    - 72 °C, 2 m
- 1 cycle:
    - 72 °C, 5 m
- Hold at 12 °C (and move to 4 °C fridge)

Column purification

Purify each sample with the Qiagen QIAquick PCR Purification Kit. Elute in 50 μl H2O. Use 1 μl eluate as template for Arb-PCR Round 2.

Semi-arbitrarily-primed PCR, Round 2

Arb-PCR, Round 2 50 μl Reaction Master (n=20)
5X GoTaq Reaction Buffer (colorless) 10.00 200
dNTP Mix, 2.5 mM each 4.00 80
primer1 (50 μM) - ARB2 0.50 10
primer2 (50 μM) - Tn-specific2 0.50 10
GoTaq DNA Polymerase (5u/μl) 0.25 5
H2O 33.75 675
Purified Round 1 Product 1.00 --

ARB2

- 30 cycles:
    - 94 °C, 30 s
    - 55 °C, 30 s
    - 72 °C, 1 m 30 s
- 1 cycle:
    - 72 °C, 5 m
- Hold at 12 °C (and move to 4 °C fridge)

Column purification

Purify each sample with the Qiagen QIAquick PCR Purification Kit. Elute in 50 μl H2O.

Note that even for successful samples a band is typically not observed by gel electrophoresis.

Sequencing

Check by Nanodrop. Yield should be 5-20 ng/ul; concentrate with a DNA Speedvac if concentration <5 ng/μl, and submit for DNA sequencing.

Scaling Up

For large numbers of samples, consider the following changes to scale up the analysis:

  • Use colonies or overnight culture diluted (1:100) in water, instead of gDNA prep.
  • Do 10 μl PCR reactions.
  • Use EXOSAP-IT in place of each column purification. This inactivates the previous round primers, so removing them is no longer necessary. Add 4 μl EXOSAP-IT to the Round 1 products and incubate. Then dilute the sample to 100 μl and use 1 μl of that sample in the Round 2 Arb reaction. Add 4 μl EXOSAP-IT to the Round 2 product and incubate. Then add sequencing primer and submit.

EXOSAP-IT incubation

- 1 cycle:
    - 37 °C, 15 m
    - 80 °C, 15 m
- Hold at 12 °C (and move to 4 °C fridge)