John M. S. Bartlett.pdf - Bio-Nica.info

Serial Analysis of Gene Expression 279
3.13. Cloning Concatemers
Clone concatemers using the Zero Background Cloning Kit. The pZErO ® -1 vector
contains a lethal gene which is disrupted by insertion of DNA, thus only positive
recombinants should grow (this is the theory: in practice, some colonies do lack inserts).
1. To linearize the vector, mix 1 µL of pZErO ® -1 (1 µg/µL), 7 µL of dH 2 O, 1 µL of NEBuffer
2 and 1 µL of SphI. Incubate at 37°C for 15 to 30 min (not over 30 min).
2. P/C extract and ethanol precipitate. Resuspend in 30 µL of LoTE.
3. To 1 µL of SphI-linearized pZErO ® , add the 6 µL of purified concatemers, 1 µL of
10× ligase buffer and 1 µL of T4 DNA Ligase. Include no insert (omit concatemers) and no
ligase (omit concatemers and ligase) controls. Incubate at 16°C for 2 h.
4. P/C extract and ethanol precipitate. Resuspend in 6 µL of LoTE.
5. Transfect 2 µL of DNA into ELECTROMAX DH10B Escherichia coli cells by electroporation,
according to manufacturer’s instructions.
6. Plate one-tenth of transfected bacteria onto each 13-cm Zeocin -containing low-salt LB
agar plate. Keep all plates at 4°C until inserts are checked. If inserts are of appropriate
size, plates may be used for large-scale sequencing.
3.14. Screening of Transformants by PCR
to Identify Long Concatemer Inserts
Perform PCR with vector-specific primers to determine insert size in each bacterial
colony. Tubes (0.5 mL) may be used initially, but 96-well PCR plates are more efficient
for large-scale screening. The 25-µL reaction volume can be reduced, for example,
to 16 µL.
1. Step 1, option 1: Johns Hopkins protocol. Per 25 µL of reaction, use: 2.5 µL of 10× SAGE
PCR buffer; 1.25 µL of DMSO; 1.25 µL of 10 mM dNTPs; 0.5 µL of each of M13 forward
and reverse Primers; 19 µL of dH 2 O; and 0.2 µL of PLATINUM Taq DNA Polymerase. The
cycling parameters are 95°C for 2 min; 25 cycles of 95°C for 30 s, 56°C for 1 min and
70°C for 30 s; then 70°C for 5 min. Step 1, option 2: own modification. The colony PCRs
are robust and work well with the Taq PCR core kit. Per 100 µL of reaction volume, use:
61.5 µL of dH 2 O, 10 µL of 10× Qiagen PCR buffer, 5 µL of 25 mM MgCl 2 , 20 µL of
5× Q-Solution, 2 µL of 10 mM dNTPs, 0.5 µL of each of M13 forward and reverse Primers,
and 0.5 µL of Taq DNA Polymerase. The cycling parameters are 94.5°C for 1.5 min;
30 cycles of 94.5°C for 30 s, 52°C for 1 min and 72°C for 1 min; then 72°C for 5 min.
2. Touch a single colony with a new pipet tip then dip into reaction mix and shake. Repeat
as necessary. Perform PCR.
3. Run 5 µL of each reaction on a 2% agarose gel with a 100-bp ladder.
3.15. Sequencing of SAGE Concatemer Inserts
Select and sequence the PCR products of 500 bp in size or over because these
should contain at least 15 tags (226 bp of flanking pZErO ® -1 vector plus 12 to 13 bp
per tag).
1. Before sequencing, purify the PCR product (partly to remove primers because M13
forward is used again). Individual phenol chloroform extraction and ethanol precipitation
is one option. However, Qiagen’s QIAquick 8 PCR Purification Kit with the QIAvac 6S
are more efficient for large-scale sequencing (see Note 7).
2. Sequence according to local preference. For example, use the BigDye Primer Kit with
one-half to one-tenth of the purified PCR product per sequencing reaction and the M13