Ligate cDNA to Vector:
6 µl cDNA (1 µg)
2.2 µl gt11 (1 µg)
1 µl 10X ligation buffer
1 µl T4 ligase (1 Unit @ 1 U/µl)
Incubate overnight at 15°C, package and plate, or transform, and then screen. Using this protocol, we obtained about 100,000 pfu per µg of packaged DNA).
Notes on Reagents
- Me(Hg)OH
- DANGEROUS, store in fume hood
- RNAsin
- RNAse inhibitor (Promega Biotek, 30 U/µl)
- Oligo-dT
- Primer for first strand (Collaborative Research, dT 12-18; stock 1 mg/ml in H2O; store -20°C or -70°C)
- RTase
- Reverse transcriptase, store at -70°C (Molecular Genetics Resources, Tampa,Florida); we have also used Life Sciences RTase and obtained about 10% first-strand synthesis efficiency at 140mM KCl, and a bit lower at 80 mM KCl.
- BNAD
- beta-NAD (Sigma, mw=663.4)
- RNAseH
- from E. coli (Boehringer Mannheim Biochemicals)
- DNA Pol-I
- DNA polymerase I from E. coli
- E. coli DNA Ligase
- For second strand synthesis, specified by Okayama and Berg (New England Biolabs)
- T4 DNA Polymerase
- Used to cleave 3' overhanging ends
- EcoRI Linker
- Phosphorylated; Collaborative Research
- T4 DNA Ligase
- from IBI
