Experiments
Phosphorylation (20μM) [Click To show The Details]
DNA samples
PNK
ATP
H2O
buffer：500mM Tris-HCl(pH7.6 at 25℃)、100mM MgCl2、50mM DTT、1mM spermidine.
Preparation of CT1 /CS2 (1μM) [Click To show The Details]
Phosphorylated sample (20μM)
T4 ligase
H2O
buffer：400mM Tris-HCl、100mM MgCl2、100mM DTT、5mM ATP(pH7.8 at 25℃)
Preparation of ET1/ ES2 (0.75μM) [Click To show The Details]
CT1/CS2 (1μM)
Exo I
H2O
buffer：670mM glycine-KOH(pH9.5 at 25℃)、67mM MgCl2、10mM DTT
Double circle DNA cyclization [Click To show The Details]
ET1/ ES2(0.75μM)
S1P1、S1P2/T2P1、T2P2
T4 ligase
H2O
buffer：400mM Tris-HCl、100mM MgCl2、100mM DTT、5mM ATP(pH7.8 at 25℃)
Double circle DNA digestion [Click To show The Details]
Cyclized DNA sample (see protocol cyclization)
Exo I
Exo III
buffer1：670mM glycine-KOH (pH9.5 at 25℃)、67mM MgCl2、10mM DTT
buffer2：660mM Tris-HCl (pH8.0 at 30℃)、6.6mM MgCl2
Transcription [Click To show The Details]
digested DNA sample (see protocol digestion)
NTP
T7 RNA polymerase
H2O
RNA Degradation inhibitor
Z22/ZBP1(Added in Transcriptional regulation experiment)
buffer：200mM Tris-HCl(pH8.0)、40mM MgCl2、10mM spermidine -(HCl)3、125mM NaCl
