2 Enzymatic Modification of DNA and RNA
49
activity, follow the safety regulations of your laboratory and discard pipet
tips and contaminated solutions properly.
6. Incubate at 37°C for 1 hour.
7. Add 1 ).11 RNasin™ and 1 ).11 DNase I. Mix gently.
8. Incubate at 37°C for 10 min.
9. Purify the mixture by using a guanidine isothiocyanate/silica-gel spin
column system (e. g. RNeasy™, Qiagen).
10. Elute the RNA in 20-100 ).11 DEPC treated water. Determine the counts
per minute in 1 ).11 ifthe RNA is radiolabeled. Check 1 ).11 in a denaturing
polyacrylamide or agarase gel (section 6).
Note: For some applications, it may be sufficient to remove unincorporated
nucleotides and small molecules by small scale gel filtration (e. g. QuickSpin®, Boehringer). If the RNA is not immediately used, it should be stored
under ethanol at -70°C. Add 2 volumes of ethanol. Torecover the RNA, add
0.1 volume ammonium acetate 5 M, mix, store at -20°C for 30 min, and
centrifuge in a microfuge for 10 min. at 12000 rpm at 4°C.
Subprotocol 9
Terminal Deoxynucleotidyl Transferase (TdT)
TdT is a DNA polymerase which does not copy a template. lt adds nucleotides preferentiallyto the 3' termini of existing DNA molecules. In vivo, TdT
is found only in prelymphocytes and during early stages of lymphoid differentiation. In vitro, the enzyme is used to add homopolymerk tails to
vector and cDNA, and to 3' end-labei DNA.
Procedure
For a protocol, see manufactures recommendations.
References
McClarin JA, Frederick BC, Wang P, Greene H, Bryer W, Grable J, Rosenberg JM (1986).
Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution.
Science 234: 1526-1541.
49
activity, follow the safety regulations of your laboratory and discard pipet
tips and contaminated solutions properly.
6. Incubate at 37°C for 1 hour.
7. Add 1 ).11 RNasin™ and 1 ).11 DNase I. Mix gently.
8. Incubate at 37°C for 10 min.
9. Purify the mixture by using a guanidine isothiocyanate/silica-gel spin
column system (e. g. RNeasy™, Qiagen).
10. Elute the RNA in 20-100 ).11 DEPC treated water. Determine the counts
per minute in 1 ).11 ifthe RNA is radiolabeled. Check 1 ).11 in a denaturing
polyacrylamide or agarase gel (section 6).
Note: For some applications, it may be sufficient to remove unincorporated
nucleotides and small molecules by small scale gel filtration (e. g. QuickSpin®, Boehringer). If the RNA is not immediately used, it should be stored
under ethanol at -70°C. Add 2 volumes of ethanol. Torecover the RNA, add
0.1 volume ammonium acetate 5 M, mix, store at -20°C for 30 min, and
centrifuge in a microfuge for 10 min. at 12000 rpm at 4°C.
Subprotocol 9
Terminal Deoxynucleotidyl Transferase (TdT)
TdT is a DNA polymerase which does not copy a template. lt adds nucleotides preferentiallyto the 3' termini of existing DNA molecules. In vivo, TdT
is found only in prelymphocytes and during early stages of lymphoid differentiation. In vitro, the enzyme is used to add homopolymerk tails to
vector and cDNA, and to 3' end-labei DNA.
Procedure
For a protocol, see manufactures recommendations.
References
McClarin JA, Frederick BC, Wang P, Greene H, Bryer W, Grable J, Rosenberg JM (1986).
Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution.
Science 234: 1526-1541.