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

cDNA Libraries from Few Cells 505
10. 10× PCR buffer (see Chapter 1).
11. 42°C incubator.
2.9. Blunt-End Cloning of PCR Products
1. Unpurified PCR products.
2. T4 DNA polymerase (4 U/µL, Amersham).
3. Agarose gel (choose agarose concentration according to the PCR product length).
4. QIAEX II purification kit (Qiagen, Inc., Chatsworth, CA).
5. ATP (3 and 8 mM ).
6. T4 polynucleotide kinase 5 U/µL (Amersham).
7. 10× T4 polynucleotide kinase buffer (Amersham).
8. Dephosphorylated SmaI pUC19 vector (Appligene).
9. T4 DNA ligase, 4 U/µL.
10. Electrocompetent XLI blue cells.
11. Escherichia coli electroporation equipment.
2.10. Direct PCR on Colonies
1. Inoculating needles.
2. PCR reagent, including primers. With pUC vectors, use M13 universal sequencing primer
and M13 reverse sequence primer.
3. Methods
3.1. RNA Extraction
All manipulations must be performed in an RNase-free environment and with PCR
anticontamination material. In our hands, the best extraction yield for low amount of
material is obtained with the RNAZol kit. When working with tissues, use a polytronR
TP1200 to homogenize in the RNAzol solution.
Follow the supplier’s instructions with two important modifications.
1. Add 1 µL of DT40 (5 mg/mL) in the RNAZol solution prior to the homogenization step.
This will increase the extraction yield.
2. At the end, resuspend the pellet in 20 µL of ddH 2 O and store at –80°C.
If the extraction of polyA + RNA is required, use the Dynabeads mRNA purification
kit (Dynal); do not use the Dynabeads mRNA DIRECT kit. Elute from the magnetic
beads with 20 µL of ddH 2 O instead of elution buffer and store at –80°C.
3.2. Synthesis of the ss-cDNA (see Note 2)
The final volume for the reverse transcription is 50 µL.
3.2.1. AMV RTase Preincubation Mix
1. Add, on ice, in a sequential manner the following reagents: 1.5 µL of H 2 O, 3 µL of 10× FSB,
2.5 µL of 0.1 M DTT, 3 µL of 10 mM each dNTP (lithium free), 1 µL of 5 mg/mL BSA,
5 µL [α 32 P] dATP (100 U/µL) (3000 Ci/mmol), 1 µL of RNasin (36 U/µL), 10 µL of
20 mM PPi, and 1 µL of AMV RTase.
2. Preincubate on ice for 30 min.