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

Serial Analysis of Gene Expression 277
3.7. Blunt-Ending-Released cDNA Tags
1. To each of the two tubes (from linkers 1 and 2) containing released cDNA tags, add 31 µL
dH 2 O, 5 µL of 10× EcoPol buffer, 0.5 µL of 100× BSA, 2.5 µL of 10 mM dNTPs, and
1 µL of DNA polymerase I large fragment (Klenow).
2. Incubate at 37°C for 30 min, and then pool both tubes of blunt-ended tags.
3. P/C extract, ethanol precipitate, and resuspend in 12 µL of LoTE.
3.8. Ligating Blunt-Ended Tags to Form 102-bp Ditags
1. Set up two new tubes, ideally of small size (0.2 mL). One tube is for the ditag ligation
reaction. The other is a negative ligation control, to exclude cross-contamination at the
next, PCR, step: set this tube up first. To each of the two tubes, add 4 µL of blunt-ended
tags, 0.8 µL dH 2 O and 0.6 µL of 10× ligase buffer.
2. To the negative control tube, add 0.6 µL of dH 2 O.
3. To the ditag reaction, add 0.6 µL of T4 DNA ligase.
4. Cover with a drop of mineral oil to avoid evaporation and incubate at 16°C overnight.
3.9. PCR Amplification of 102-bp Ditags
The PCR aims to produce sufficient 102-bp ditag DNA for subsequent isolation and
concatemerization of 26 bp ditags but may itself be problematic (see Note 8).
1. After ligation, add 14 µL of LoTE to increase volume to 20 µL and mix. Take 1 µL
and dilute 100-fold with LoTE. Use 1 µL of the dilution in a 50- or 100-µL PCR with
biotinylated SAGE Primers 1 and 2 (11). To avoid cross-contamination, set up the two
negative control reactions (no template and no ligase) first.
2. Step 2, option 1: Johns Hopkins protocol. Per 50-µL reaction, use 30.5 µL dH 2 O, 5 µL
of 10× SAGE PCR buffer, 3 µL of DMSO, 7.5 µL of 10mM dNTPs, 1 µL of each of
SAGE Primers 1 and 2, and 1 µL PLATINUM Taq DNA Polymerase. The cycling parameters,
optimized for a Hybaid thermal cycler, are as follows: 94°C for 1 min; 26 to 30 cycles of
94°C for 30 s, 55°C for 1 min and 70°C for 1 min; then 70°C for 5 min. Optimize with
different template dilutions (1/50, 1/100, or 1/200 per reaction). Step 2, option 2: own
modification. The HotStarTaq DNA Polymerase Kit routinely works well with 1 µL, or
often less, of the 1/100 dilution with no need for further adjustment of template dilution.
Per 100-µL reaction, use: 60.5 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 SAGE
Primers 1 and 2, and 0.5 µLHotStarTaq DNA Polymerase. The cycling parameters are:
94.5°C for 15 min; 26 to 30 cycles of 94.5°C for 30 s; 56°C for 1 min and 72°C for 1 min;
then 72°C for 5 min.
3. Optimize the cycle numbers between 26 and 30. More than 30 cycles usually results in
high molecular weight smearing with less of the desired product.
4. After PCR, load 10 µL of each reaction on a 12% polyacrylamide gel with a 20-bp ladder.
Run at 160 V for 2.5 h until the bromophenol blue dye front has run out of the gel and
the xylene cyanol is 1 to 2 cm from the bottom, then stain (see Note 9). The amplified
ditags should produce a 102-bp band. Background bands are common: the brightest runs
at 80 bp and contains amplified ligated linkers without tags. The negative controls should
contain no product.
5. After optimization, perform large-scale PCR by preparing then distributing a master-mix
into three 96-well PCR plates with 50 or 100 µL in each well.
6. After PCR, pool the reactions. P/C extract and ethanol precipitate, scaling up as needed.
The large volumes can be dealt with using either multiple 1.5-mL microcentrifuge tubes or