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Megaprimer RF Cloning Protocol

Megaprimer restriction-free (RF) cloning can be used to insert DNA fragments like PCR products or gBlocks Gene Fragments into plasmids without restriction enzymes or ligation. The procedure involves designing the insert with 30-80 bp overlaps to the vector and using PCR with a high-fidelity polymerase like Phusion to incorporate the insert. Then a DpnI digestion removes the original plasmid template before transformation into E. coli, where the insert is integrated into the plasmid with nicks in each strand sealed by the cell's machinery. gBlocks Gene Fragments are double-stranded DNA fragments of varying lengths that can be used as the insert for this cloning method.

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207 views1 page

Megaprimer RF Cloning Protocol

Megaprimer restriction-free (RF) cloning can be used to insert DNA fragments like PCR products or gBlocks Gene Fragments into plasmids without restriction enzymes or ligation. The procedure involves designing the insert with 30-80 bp overlaps to the vector and using PCR with a high-fidelity polymerase like Phusion to incorporate the insert. Then a DpnI digestion removes the original plasmid template before transformation into E. coli, where the insert is integrated into the plasmid with nicks in each strand sealed by the cell's machinery. gBlocks Gene Fragments are double-stranded DNA fragments of varying lengths that can be used as the insert for this cloning method.

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gBlocks Gene Fragments

Protocol: Megaprimer RF Cloning


Megaprimer restriction-free (RF) cloning uses PCR methods to assemble a
double-stranded insert such as a PCR product or gBlocks Gene Fragments into any position, in any plasmid vector, without the need for
restriction and ligation (Figure 1). A high-fidelity DNA polymerase such
as the Phusion DNA Polymerase (www.NEB.com/Phusion) is used in this
protocol to limit the introduction of sequence errors [1].

gBlocks Gene Fragments

Resuspending your gBlocks Gene Fragments


The dried down gBlocks Gene Fragment pellet can become displaced
from the bottom of the tube during shipping.
Centrifuge the tube for 35 sec at a minimum of 3000 x g to pellet
the material to the bottom of the tube.

50 L RF Reaction
Nuclease-free H2O

Adjust to final 50 L
10 L

10 mM dNTPs

1 L

Cloning plasmid

20 ng

gBlocks Gene Fragments

100 ng

Phusion DNA Polymerase

0.8 L

2. Gently mix the reaction and spin down in a microcentrifuge.


3. Carry out the RF cloning reaction in a thermal cycler with a heated lid.

Cycling conditions

Briefly vortex and centrifuge

The table shows general guidelines for cloning a gBlocks Gene Fragment into a
cloning vector, using the megaprimer RF cloninng procedure.

Resuspension volume of TE buffer (L)


for gBlocks Fragments synthesis scales
250 ng

500 ng

1000 ng

10 ng/L

25

50

100

20 ng/L

Not
recommended

25

50

50 ng/L

Not
recommended

10

20

Cycling Parameters
Step

Storing your gBlocks Gene Fragments


gBlocks Gene Fragments can be stored in TE at 20C for up to 24
months. If gBlocks Gene Fragments will be stored for less than 1 month,
they can be resuspended in nuclease-free water instead of TE.

Required materials

1. Set up the amplification reaction on icereaction components for a


50 L reaction are shown.

Add TE to the tube for your desired final concentration

Final
concentration

Megaprimer RF cloning procedure

5X Phusion HF or GC buffer

gBlocks Gene Fragments are chemically synthesized, double-stranded


DNA, delivered normalized to 250, 500, or 1000 ng, depending on length,
and dried down. Order at www.idtdna.com/gblocks.

INTEGRATED DNA TECHNOLOGIES

gBlocks Gene Fragments with 3080 bp 3' and 5', insertion site
overlaps (Figure 1)
Amplification primers

Cycles

Temperature

Time

Initial denaturation

95C

30 sec

Denaturation
Annealing
Extension

25

95C
60C
72C

30 sec
1 min
5 min

Final extension

72C

7 min

Hold

4C

DpnI Digestion of methylated, empty vector


Remove empty, target-vector template with DpnI digest.
50 L DpnI Rxn
Completed RF cloning reaction

10 L Aliquot

DpnI (20U/ L)

1 L

Phusion DNA Polymerase (www.NEB.com/Phusion)

DpnI buffer (10X)

5L

DpnI enzyme and buffer

BSA

0.5 L

Competent cells and transformation reagents

ddH2O

33.5 L

G
A

B
B

1. Incubate the reaction for 12 hr at 37C.


A

2. Transform directly into E. coli.

Reference

Figure 1. Overview of Megaprimer RF Cloning. A double-stranded,


DNA gBlocks Gene fragment or PCR product is designed with
3080 bp overlap at the 3' and 5' ends and the vector insertion site
(A and B). Using the RF protocol, the fragment is incorporated using
PCR methods and a high-fidelity polymerase, as described. The
finished plasmid contains the cloned gene (G) with a nick on each
strand that is sealed by endogenous E. coli mechanisms.

1. Unger T, Jacobovitch Y, et. al. (2010) Applications of the Restriction


Free (RF) cloning procedure for molecular manipulations and protein
expression. J Struct Biol. 172(1). 3444.

PHUSION is a registered trademark and property of Thermo Fisher Scientific.

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