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Digestion of PCR Products

• Cleavage of PCR Products Directly After Amplification
• Cleavage Efficiency Close to the Termini of PCR Fragments (see below)

Cleavage Efficiency Close to the Termini of PCR Fragments

Some restriction enzymes cleave DNA poorly when their recognition sites are located near the end of a DNA strand. The Table below presents activities of Fermentas restriction enzymes when their target sites are located close to the end of a PCR product.

Experiments were performed as follows:

PCR primers were designed with 1-5 extra nucleotides at their 5’-end adjacent to the recognition site for the restriction enzyme. The 5’-end was labeled with [32P] by T4 Polynucleotide Kinase and these labeled primers were used in the PCR reaction. PCR products were purified with the DNA Gel Extraction Kit, and precipitated with ethanol. DNA aliquots (0.5 µg) were incubated with 10 units of restriction enzymes in its optimal buffer (40 µl) for 1 hour at the recommended temperature. Reaction products were separated on 10% PAGE and the percentage of their cleavage was determined using OptiQuant Image Analysis software.

Table. Cleavage Efficiency Close to the Termini of PCR Fragments

	Cleavage efficiency:
	- 0%
	- 0-20%
	- 20-50%
	- 50-100%
*	- incubation was performed for 16 hours

Enzyme	Base pairs from site to fragment end
	1	2	3	4	5
AarI	20-50		50-100
AasI	50-100
AatII	0	0-20	20-50	50-100
Acc65I	0-20	50-100
AdeI	50-100
AjiI	50-100
AluI	0-20	20-50	50-100
Alw21I	50-100
Alw26I	50-100
Alw44I	0	20-50	50-100
ApaI	50-100
BamHI	50-100
BauI	0-20	20-50	50-100
BcnI	20-50		50-100
BclI	0	50-100
BcuI	50-100
BfiI	50-100
BfmI	50-100
BfuI	50-100
BglI	20-50	50-100
BglII	0	50-100
Bme1390I	20-50		50-100
BoxI	0	50-100
BpiI	50-100
Bpu10I	20-50	50-100
Bpu1102I	50-100
BseDI	0	50-100
BseGI	50-100
BseJI	0	50-100
BseLI	0	50-100
BseMI	0-20	50-100
BseMII	50-100
BseNI	0	50-100
BseSI	50-100
BseXI	20-50	50-100
Bsh1236I	50-100
Bsh1285I	0-20	50-100
BshNI	50-100
BshTI	20-50	50-100
Bsp68I	0	50-100
Bsp119I	50-100
Bsp120I	20-50	50-100
Bsp143I	50-100
Bsp1407I	20-50	50-100
BspLI	50-100
BspPI	0	50-100
BspTI	0	0-20	50-100
Bst1107I	0-20	50-100
BstXI	0		50-100
Bsu15I	50-100
BsuRI	0-20	20-50	50-100
BveI	0-20		50-100
Enzyme	Base pairs from site to fragment end
	1	2	3	4	5
CaiI	0	0-20	50-100
CfrI	0	50-100
Cfr9I	20-50	50-100
Cfr10I	20-50	50-100
Cfr13I	50-100
Cfr42I	50-100
CpoI	50-100
CseI	50-100
Csp6I	50-100
DpnI	50-100
DraI	0	0-20	50-100
Eam1104I	0	50-100
Eam1105I	0	50-100
Ecl136II	50-100
Eco24I	50-100
Eco31I	20-50		50-100
Eco32I	20-50	50-100
Eco47I	50-100
Eco47III	0	0-20	50-100
Eco52I	0-20	50-100
Eco57I	50-100
Eco57MI	50-100
Eco72I	0-20	50-100
Eco81I	50-100
Eco88I	50-100
Eco91I	20-50	50-100
Eco105I	20-50	50-100
Eco130I	0		50-100
Eco147I	0	50-100
EcoO109I	50-100
EcoRI	50-100
EcoRII	0	0-20	20-50	50-100
EheI	20-50		50-100
Esp3I	50-100
Enzyme	Base pairs from site to fragment end
	1	2	3	4	5
FaqI	0-20	50-100
FspAI	0-20	20-50	50-100
FspBI	20-50	50-100
GsuI	50-100
HhaI	50-100
Hin1I	50-100
Hin1II	0	0-20	50-100
Hin6I	0		0-20	50-100
HincII	50-100
HindIII	0	0-20	50-100
HinfI	50-100
HpaII	0-20	50-100
HphI	0	50-100
Hpy8I	0-20	50-100
HpyF3I	20-50	50-100
HpyF10VI	50-100
KpnI	50-100
Kpn2I	0	50-100
KspAI	20-50	50-100
LguI	50-100
LweI	20-50	50-100
MbiI	0	0-20	50-100
MboI	50-100
MboII	50-100
MlsI	0-20	50-100
MluI	20-50		50-100
MnlI	0	50-100
Mph1103I	20-50	50-100
MspI	0-20	50-100
MssI	20-50	50-100
MunI	20-50	50-100
MvaI	0	20-50	50-100
Mva1269I	0	50-100
NcoI	0	50-100
NdeI*	0-20	20-50	50-100
NheI	0	20-50		50-100
NmuCI	20-50	50-100
NotI	20-50	50-100
NsbI	0	0-20	50-100
Enzyme	Base pairs from site to fragment end
	1	2	3	4	5
OliI	0-20	20-50	50-100
PaeI	0	0-20		20-50	50-100
PagI	20-50	50-100
PasI	50-100
PauI	0	50-100
PdiI	0-20	50-100
PdmI	50-100
PfeI	50-100
Pfl23II	0-20	20-50	50-100
PfoI	0	20-50	50-100
Ppu21I	50-100
PscI	0	50-100
Psp5II	0	50-100
Psp1406I	0-20	50-100
PsuI	0-20	50-100
PstI	0-20	50-100
PsyI	0	50-100
PvuI	20-50		50-100
PvuII	50-100
RsaI	50-100
SacI	50-100
SalI	20-50		50-100
SatI	0	50-100
ScaI	0-20	50-100
SchI	50-100
SdaI	0-20	50-100
SduI	50-100
SfiI	50-100
SgsI	50-100
SmaI	50-100
SmiI	0-20	50-100
SmoI	0	50-100
SmuI	50-100
SsiI	50-100
SspI	0-20	50-100
TaaI	20-50	50-100
TaiI	50-100
TaqI	0	20-50		50-100
TasI	0	20-50	50-100
TatI	0-20		20-50	50-100
TauI	20-50	50-100
Tru1I	0	0-20		50-100
TsoI	20-50	50-100
Van91I	0	50-100
VspI	50-100
XagI	20-50	50-100
XapI	20-50		50-100
XbaI	20-50	50-100
XceI	0	50-100
XhoI	0-20	50-100
XmaJI	50-100
XmiI	50-100
I-SceI	50-100

catalog@fermentas.com

Updated vasario 01, 2008 09:29