Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site
Double digestions within multiple cloning sites (MCS) are often ineffective when the DNA target sequences are in close vicinity, or they are too close to the end of a DNA molecule (see Table "Cleavage Efficiency Close to the Termini of PCR Fragments"). Nevertheless, it is often necessary to perform effective double digestions within the cloning sites in which restriction targets are in close vicinity. Experimental guidelines for these applications are presented in the table below. The data were generated using a linearized pUC19 plasmid. The plasmid was initially cleaved with a primary restriction enzyme (the first cut), and was then end-labeled with [32P] by T4 Polynucleotide Kinase. This DNA was digested for one hour with varying amounts (2, 5 and 10 units) of a second restriction enzyme in an optimal reaction buffer (the second cut). Reaction products were separated by PAGE, and the amount of the label left on the DNA was determined by autoradiography. A decrease in radioactivity reflects the cleavage by the second restriction enzyme. The results presented in the table below should be used to choose the optimal order of DNA digestions.
Note
Thus, the first reaction should be performed with a restriction enzyme that cleaves inefficiently close to the end of DNA, while the second digestion should be performed with a restriction enzyme which tolerates a close proximity to the DNA end.Table. Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site.
* Overhangs are not included, only double-stranded portion of the DNA.
Enzyme pair First cut Second cut Efficiency of
the second cut, %Base pairs from
the first cut*Acc65I/BamHI Acc65I BamHI 100 4 BamHI Acc65I 100 4 Acc65I/Ecl136II Acc65I Ecl136II 100 1 Ecl136II Acc65I 50-100 3 Acc65I/Eco24I Acc65I Eco24I 100 1 Eco24I Acc65I 0 1 Acc65I/EcoRI Acc65I EcoRI 100 7 EcoRI Acc65I 100 7 Acc65I/SacI Acc65I SacI 50-100 1 SacI Acc65I 0-20 1 Acc65I/XapI Acc65I XapI 50-100 7 XapI Acc65I 50-100 7 BamHI/Cfr9I BamHI Cfr9I 50-100 0 Cfr9I BamHI 50-100 0 BamHI/Eco88I BamHI Eco88I 100 0 Eco88I BamHI 50-100 0 BamHI/HincII BamHI HincII 50-100 7 HincII BamHI 100 9 BamHI/KpnI BamHI KpnI 100 4 KpnI BamHI 100 4 BamHI/SalI BamHI SalI 50-100 7 SalI BamHI 100 7 BamHI/XbaI BamHI XbaI 20-50 1 XbaI BamHI 100 1 Cfr9I/Ecl136II Cfr9I Ecl136II 50-100 5 Ecl136II Cfr9I 100 7 Cfr9I/Eco24I Cfr9I Eco24I 50-100 5 Eco24I Cfr9I 100 5 Cfr9I/SacI Cfr9I SacI 50-100 5 SacI Cfr9I 50-100 5 Cfr9I/XbaI Cfr9I XbaI 50-100 6 XbaI Cfr9I 50-100 6 Ecl136II/Eco88I Ecl136II Eco88I 100 7 Eco88I Ecl136II 50-100 5 Ecl136II/EcoRI Ecl136II EcoRI 100 3 EcoRI Ecl136II 100 1 Ecl136II/XapI Ecl136II XapI 50-100 3 XapI Ecl136II 50-100 1 Eco24I/Eco88I Eco24I Eco88I 100 5 Eco88I Eco24I 50-100 5 Eco24I/EcoRI Eco24I EcoRI 100 1 EcoRI Eco24I 100 1 Eco24I/KpnI Eco24I KpnI 50-100 1 KpnI Eco24I 0 1 Eco24I/XapI Eco24I XapI 50-100 1 XapI Eco24I 20-50 1 Eco88I/SacI Eco88I SacI 100 5 SacI Eco88I 50-100 5 Eco88I/XbaI Eco88I XbaI 100 6 XbaI Eco88I 100 6 EcoRI/Cfr9I EcoRI Cfr9I 50-100 11 Cfr9I EcoRI 100 11 EcoRI/Eco88I EcoRI Eco88I 50-100 11 Eco88I EcoRI 100 11 EcoRI/KpnI EcoRI KpnI 100 7 KpnI EcoRI 100 7 EcoRI/SacI EcoRI SacI 50-100 1 SacI EcoRI 20-50 1 HincII/PaeI HincII PaeI 100 9 PaeI HincII 100 7 HincII/PstI HincII PstI 100 3 PstI HincII 20-50 1 HincII/SdaI HincII SdaI 0-20 2 SdaI HincII 20-50 1 HincII/XbaI HincII XbaI 50-100 3 XbaI HincII 50-100 1 HindIII/PaeI HindIII PaeI 50-100 1 PaeI HindIII 0 1 HindIII/PstI HindIII PstI 100 7 PstI HindIII 100 7 HindIII/SdaI HindIII SdaI 50-100 6 SdaI HindIII 50-100 7 KpnI/Ecl136II KpnI Ecl136II 50-100 1 Ecl136II KpnI 100 3 KpnI/SacI KpnI SacI 50-100 1 SacI KpnI 100 1 KpnI/XapI KpnI XapI 100 7 XapI KpnI 50-100 7 PaeI/PstI PaeI PstI 0 1 PstI PaeI 20-50 1 PaeI/SalI PaeI SalI 50-100 7 SalI PaeI 100 7 PaeI/SdaI PaeI SdaI 0 0 SdaI PaeI 20-50 1 PstI/BamHI PstI BamHI 50-100 13 BamHI PstI 50-100 13 PstI/SalI PstI SalI 0 1 SalI PstI 50-100 1 PstI/XbaI PstI XbaI 100 7 XbaI PstI 50-100 7 SacI/SmaI SacI SmaI 100 5 SmaI SacI 50-100 7 SacI/XapI SacI XapI 0-20 1 XapI SacI 50-100 1 SalI/SdaI SalI SdaI 0 0 SdaI SalI 20-50 1 SalI/XbaI SalI XbaI 50-100 1 XbaI SalI 0-20 1 SdaI/XbaI SdaI XbaI 100 7 XbaI SdaI 50-100 6 SmaI/Acc65I SmaI Acc65I 0-20 1 Acc65I SmaI 0 -1 SmaI/BamHI SmaI BamHI 50-100 2 BamHI SmaI 0-20 0 SmaI/Ecl136II SmaI Ecl136II 50-100 7 Ecl136II SmaI 100 7 SmaI/Eco24I SmaI Eco24I 50-100 7 Eco24I SmaI 100 5 SmaI/KpnI SmaI KpnI 0 1 KpnI SmaI 0 -1 SmaI/XbaI SmaI XbaI 50-100 8 XbaI SmaI 100 6
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Updated lapkričio 08, 2006 11:34
