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Description: Pfu-X Core Kit contains all reagents required for PCR (except template and primer) in one box combining simple handling with high flexibility. The premium quality polymerase, ultrapure dNTPs and the optimized complete reaction buffer ensure superior amplification results.
Pfu-X Polymerase is the ideal choice for applications where the efficient amplification of DNA with highest fidelity is required. The enzyme is a genetically engineered Pfu DNA polymerase, showing a 2-fold higher accuracy and an increased processivity, resulting in shorter elongation times.
The enzyme catalyzes the polymerization of nucleotides into duplex DNA in 5'→3' direction but does not possess a 5'→3' exonuclease replacement activity. Its inherent 3'→5' exonuclease proofreading activity results in a greatly increased fidelity of DNA synthesis compared to Taq polymerase.
Pfu-X Polymerase-generated PCR fragments are blunt-ended. The enzyme is highly purified and free of bacterial DNA.
25 x 10-6 The error rate (ER) of a PCR reaction is calculated using the equation ER = MF/(bp x d), where MF is the mutation frequency, bp is the number of base pairs of the fragment and d is the number of doublings (2d = amount of product / amount of template).
Component — PCR-237S — PCR-237L
Pfu-X Polymerase
2.5 units/μl
in storage buffer*
red cap — 40 μl
100 units — 200 μl
500 units
dNTP Mix
10 mM each
dATP, dCTP,
dGTP, dTTP
white cap — 100 μl — 500 μl
Pfu-X Buffer
10x conc.
green cap — 500 μl — 2 x 1.2 ml
PCR-grade Water
white cap — 2 x 1,2 ml — 2 x 6 ml
* (50 % Glycerol, 50 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 1 mM DTT 0.1 % Tween 20, 0.1 % Nonidet P-40)
5 μl — 10x Pfu-X Buffer — green cap
1 μl — dNTP Mix — white cap
0.4 μM — each Primer — -
1 - 100 ng — template DNA — -
0.5 μl
(1.25 units) — Pfu-X Pol — red cap
Fill up to 50 μl — PCR-grade water — -
Please note that it is essential to add the polymerase as last component.
Three-step standard protocol
initial
denaturation — 95 °C — 2 min — 1x
denaturation — 95 °C — 20 sec — 25-30x
annealing1) — 50 - 68 °C — 30 sec — 25-30x
elongation2) — 68 °C — 1 min/kb — 25-30x
final
elongation — 68 °C — 1 min/kb — 1x
Two-step protocol for amplification of longer fragments (>3 kb)
Please note that for performing two-step cycling a sufficiently high primer Tm is necessary. If Tm of primers is below 65 °C or two-step PCR does not yield a sufficient product quality the three-step cycling protocol is recommended.
initial
denaturation — 95 °C — 2 min — 1x
denaturation — 95 °C — 20 sec — 25-30x
annealing/
elongation1,2) — 68 °C — 30 sec/kb — 25-30x
final
elongation — 68 °C — 30 sec/kb — 1x
1)The annealing temperature depends on the melting temperature of the primers used. 2)The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.
Ligase-free Cloning is based on a cloning technique invented by Quan and Tian in 2009. It offers a number of advantages over conventional cloning methods. The system:
Ligase-free Cloning is based on generation of inserts with homologous ends to the linearized vector. In a circularization reaction, vector and insert anneal due to their homologous ends.
Using a specially selected DNA polymerase, the resulting single-stranded plasmids are recircularized. These plasmids can directly be used for transformation.
They still have two nicks each, which will be repaired by E. coli's endogenous DNA repair system and thus do not have to be ligated in vitro.
If the used restriction sites are not reconstructed after cloning and not present in the target fragment, the inactivation or purification steps can be omitted.
Make sure the vector is linearized completely to reduce background in the transformation step (chapter 5).
1.1 With enzymes that can be heat inactivated:
1.2 With enzymes that cannot be heat inactivated:
Enzyme — Pfu-X Buffer — Enzyme — Pfu-X Buffer — Enzyme — Pfu-X Buffer
ApaI — 1x — HpaI — 1x — PvuII — 1x
ApaLI — 0.9x — KpnI — 1x — SalI — 2x
AsuII — 1x — MluI — 1x — ScaI — 1x
BamHI — 1x — MspCI — 2x — SlaI — 1x
BglII — 1x — NaeI — 1x — SmaI — 1x
BseAI — 1x — NcoI — 1x — SnaBI — 1x
BssHII — 1x — NdeI — 1x — SpeI — 1x
CspAI — 1x — NheI — 1x — SphI — 2x
DraI — 1x — NotI — 1x — SseBI — 1x
EcoRI — 1x — NruI — 1x — SspI — 1x
EcoRV — 1x — PstI — 1x — SstI — 1x
HindIII — 1x — PvuI — 1x — XbaI — 1x
Example: Vector was cut with NcoI and NotI. Vector is shown in lower case, primers in capitals, with the overlap in bold. Denote the primer sequence for amplification of the insert.
Forward primer: CCTTGCCACCAGATCTGCCATGNNNNNN... ...gtgccttgccaccagatctgc ...cacggaacggtggtctagacggtac
Reverse primer: ggccgccctcctcctcctttcttgttc... cgggaggaggaggaaagaacaag... ...NNNNNNCCGGCGGGAGGAGGAGGAAAGA
3.1 Standard preparation
3.2 Quick preparation
This works only if the PCR template does not have the same antibiotic resistance as the target vector, or if the PCR template is removed by Dpn I digestion.
4.1 Components
Prepare the following reaction (20 μl volume):
As a control, perform the same reaction without adding any insert.
4.2 Reaction conditions
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