- Primers
- DNA Preparation
- Semi-arbitrarily-primed PCR, Round 1
- Column purification
- Semi-arbitrarily-primed PCR, Round 2
- Column purification
- Sequencing
- Scaling Up
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.
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).
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.
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)
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.
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)
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.
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.
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.
- 1 cycle:
- 37 °C, 15 m
- 80 °C, 15 m
- Hold at 12 °C (and move to 4 °C fridge)