Vitro-RecTM PCR Cloning Kit
Technical Manual No. 0279
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Description….…………………………………………………….……………….………….
Contents….………………………………………………………………………….………..
Applications …………………………………………………………………………………..
Key Features…………………….…………………………………………………………...
Storage…………………………..…………………………………………….………………
General protocol using Vitro-RecTM PCR Cloning Kit……………………………………
Examples….…………………………………………………………………………………
Troubleshooting……………………………………….. ……...….………………………..
Ordering Information ………….…………………………………………………………..
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DESCRIPTION
The GenScript Vitro-RecTM PCR Cloning Kit is designed for the quick cloning of PCR products
without any need for restriction enzymes or ligases. The Vitro-RecTM cloning method is universal—it
works with any insert and any vector at any restriction site.
this kit rapidly generates precise, directional constructs.
Using our proprietary Vitro-RecTM Enzyme,
All that is required is 30-minute incubation at
room temperature.
II. CONTENTS
Components
Vitro-RecTM Enzyme (5U/µl)
10XVitro-RecTM Reaction Buffer
pUC57 Linearized (EcoR V,100ng/µl)
1-kb Control Insert (100ng/µl)
L00339
20 µl
50 µl
10 µl
10 µl
III. APPLICATIONS
The GenScript’s Vitro-RecTM PCR Cloning Kit can be used in a variety of applications:
 PCR cloning
 HTP PCR cloning
 Joining of DNA fragments
IV. KEY FEATURES
The Vitro-RecTM PCR Cloning Kit offers users the flexibility of quick directional cloning:
 Clones into any vector without any need for restriction enzymes, phosphatase treatments, or
ligases
 Provides up to 12 kb PCR cloning at any restriction site
 Rapidly generates precise, correctly oriented constructs and inserts
 Works with PCR that employ any thermostable polymerases
V. STORAGE
Store the kit at -20°C.
It will remain stable for at least one year, if stored properly.
VI. GENERAL PROTOCOL USING Vitro-RecTM PCR CLONING KIT
PLEASE READ ENTIRE PROTOCOL BEFORE STARTING.
A. Primer Design
If you are cloning your fragment into a linearized vector, then your primers should share the
necessary 15 bases of sequence homology with the cloning vectors on either side of the point of
insertion.
Forward Primer
5’-NNNNNNNNNNNNNNN
Vector ends
Vector
ends with
with
5’ overhang
5’
overhang
append with your specific sequence
5’-···NNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNN···-3’
3’-···NNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNN···-5’
NNNNNNNNNNNNNNN-5’
Reverse Primer
Forward Primer
5’-NNNNNNNNNNNNNNN
Vector
ends with
with
Vector ends
Blunt
overhang
blunt overhang
5’-···NNNNNNNNNNNNNNN NNNNNNNNNNNNNNN···-3’
3’-···NNNNNNNNNNNNNNN NNNNNNNNNNNNNNN···-5’
NNNNNNNNNNNNNNN-5’
Reverse Primer
Forward Primer
5’-NNNNNNNNNNNNNNN
Vector
Vectorends
endswith
with
3’
overhang
3’ overhang
5’-···NNNNNNNNNNNNNNN NNNNNNNNNNN···-3’
3’-···NNNNNNNNNNN NNNNNNNNNNNNNNN···-5’
NNNNNNNNNNNNNNN-5’
Reverse Primer
B. Preparation of Linearized Vector
To achieve a successful Vitro-RecTM PCR cloning reaction, you must first generate a very pure
preparation of linearized vector (with a very low background of uncut vector present).
Restriction
enzymes will generate different amounts of background due to differences in cutting efficiency.
Generally speaking, two enzymes cut better than any single enzyme.
Increasing the enzyme digestion
time will also reduce the background. To clone your PCR insert into your vector, prepare a linearized
vector as follows:
1. We recommend cutting the vector with two different enzymes to reduce background, unless there is
only one site available for cloning.
Cloning vector
5 µg
Restriction enzyme
n µl
Deionized water (to 100 µl)
x µl
Total volume
100 µl
We recommend adding half of the enzyme (2.5-5 U/µg) at the beginning of the digestion and the
other half about 30 minutes later.
2. Incubate your restriction digestion for at least two hours.
It may be run overnight.
3. After digestion, purify the linearized vector using any GEL or PCR purification kit. We typically cut
with two enzymes and columns purify the linearized vector. This generally gives sufficiently low
background. However, if cutting with only one enzyme, if the enzymes are of low efficiency, or if low
background is utterly critical, then gel-purify the vector to ensure low background.
4. Check the background of your vector by transforming 50-100 ng of the linearized and purified vector
into competent cells.
C. PCR Amplification and Purification
1. In general, you may perform PCR amplifications using any thermostable polymerases. Since
primers and primer dimers are inhibitory to the GenScript Vitro-RecTM PCR cloning reaction, we
recommend using hot start PCR with a touchdown protocol to increase the specificity of the resulting
PCR products. When cycling is complete, analyze your PCR product by electrophoresis on an
agarose/EtBr gel to confirm that you have obtained a single DNA fragment and to estimate the
concentration of your PCR product.
Quantify the amount of DNA by measuring against a known
standard or molecular weight marker ladder run on the same gel.
2. PCR products must be purified for successful Vitro-RecTM cloning. The method of purification
required depends on your gel electrophoresis results.
If you observe only a single, clear band without
primer dimers on the gel corresponding to your product, then removal of unincorporated dNTPs through
a simple PCR cleanup is usually sufficient. If, however, multiple bands are observed, indicating the
presence of nonspecific contaminants, we recommend that you gel-purify your fragment of interest.
D. Recombination Procedure
1. Combine the following in a 0.5 ml eppendorf tube:
Linearized vector (100-300 ng/µl)
3 µl
Purified PCR products (100-300 ng/µl)
n µl
TM
10XVitro-Rec
Reaction Buffer
Vitro-RecTM Enzyme
1 µl
1 µl
Deionized water (to 10 µl)
x µl
Total volume
10 µl
Mix the reagents gently and spin tubes briefly to bring contents to the bottom of the tube.
2. Incubate reactions at 22oC for 30 minutes, and then transfer tubes to ice for five minutes.
3. Proceed with transformation (Section E).
If you cannot transform cells immediately, then store cloning reactions at -20oC.
E. Transformation
Materials needed:
 Water bath (42oC), optional
 SOC liquid medium
 >1 x 108 cfu/µg competent cells
Transformation Procedure
1. Thaw one vial of frozen 50 µl competent cells on ice. Tap tube gently to ensure that the cells are
suspended.
Note: Competent cells should give >1 x 108 cfu/µg.
2. Add no more than 10 µl reaction mixtures to the cells.
3. Add 1 ml to SOC liquid medium.
If not, replace with a fresh batch of cells.
Leave the tube on ice for 30 minutes.
Mix well and incubate at 37oC.
4. Heat shock the cells in water bath at 42oC for 90 seconds, and then place them directly on ice for 2-3
minutes.
5. Add 600 µl of SOC medium to the cells and then incubate on a shaker at 250 rpm at 37oC for 60
minutes.
6. Centrifuge the cell down at 4000 rpm for five minutes and remove about 550 µl of medium.
7. Mix the remainder (about 100 µl) and plate all to LB/Amp plate or other appropriate
antibiotic-containing plates.
8. Invert the plate for 30 minutes and then incubate it overnight at 37oC.
VII. EXAMPLES
PCR cloning insert DNA fragment from lambda DNA into pUC57
A: A cloning number obtained with the Vitro-RecTM PCR Cloning Kit is shown below.
1 kb
2 kb
4 kb
White blot
1200
1000
221
Positive ratio
15/16
15/16
12/16
B: A PCR screening obtained with the Vitro-RecTM PCR Cloning Kit is shown below（DNA maker
is KB ladder）.
2 kb
1 kb
4 kb
2 kb
VIII.
TROUBLESHOOTING
Use the chart below to solve and avoid common problems. To confirm that your kit is working
properly, perform control reactions.
Problem
Probable Cause
Solution
Few or no colonies
The mixture contains an
Be sure that your antibiotic plates are correct
are obtained from
incorrect antibiotic.
and fresh (less than one month old).
The bacteria are not
Check transformation efficiency.
competent.
obtain >1 x 108 cfu/µg; otherwise, use fresh
the transformation.
You should
competent cells.
The cells have been
Do not add more than 10 µl of reaction mixture
transformed with too much
to 50 µl of competent cells.
reaction mixture.
the reaction mixture inhibits the transformation.
Using too much of
The volume of reaction mixture should equal
no more than 20 percent of the competent
cells’ volume.
The cloning reaction fails,
Repeat the PCR amplification and purify the
includes inhibitory
product using a different means of purification.
contaminants in PCR product,
Alternatively, perform a phenol: chloroform
and shows low DNA
extraction on your original PCR product
concentration in the reaction,
followed by ethanol precipitation.
or the primer sequences are
Either the amount of vector or the amount of
incorrect.
PCR fragment was too low to obtain a
satisfactory reaction product.
Alternatively,
the 2:1 molar ratio of PCR fragment to linear
vector used in the Vitro-RecTM protocol may not
have been optimal. Check primer sequences
to ensure that they provide 15 bases of
homology with the region flanking the vector
cloning site (see Section IV) if using
Vitro-RecTM cloning.
Large numbers of
Your cloning vector is not
When using a linearized vector for cloning, it
colonies contained
completely linearized.
must be purified to remove any uncut vector
before use in the GenScript Vitro-RecTM PCR
no insert.
cloning reaction. Recut your vector.
It may
be necessary to gel-purify your linearized
vector.
The cloning reaction has been
If your insert is amplified from a plasmid,
contaminated by plasmids
closed circular DNA (vector) may have carried
with the same antibiotic
through the purification and contaminated the
resistance.
cloning reaction. To ensure the removal of
any plasmid contaminants, it may be
necessary to gel purify your PCR product.
Alternatively, the PCR product can be treated
with DpnI to remove the parental vector
template after PCR amplification.
Clones contain
The PCR product has been
If your PCR product is not a single distinct
incorrect insert.
contaminated with nonspecific
band, then it may be necessary to gel-purify it
sequences.
to ensure cloning of the correct insert.
IX. ORDERING INFORMATION
Vitro-RecTM PCR Cloning Kit
Cat. No. L00339.
For Research Use Only
GenScript Corporation
120 Centennial Ave., Piscataway, NJ 08854
Tel: 732-885-9188
Fax: 732-210-0262, 732-885-5878
Email: info@genscript.com
Web: www.genscript.com