[CV1100] GetClone™ PCR Cloning Vector II, 20 Rxn

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Description 

The GetClone™ PCR Cloning Vector II is a positive selection system for high efficiency cloning of blunt end DNA or amplicons. This cloning vector contains a lethal gene which can be disrupted by ligation of a blunt end DNA insert into the cloning site. Only colonies with inserted vectors are able to propagate, eliminating the additional needs of IPTG and X-Gal for blue/white screening. This cloning vector includes ampicillin and kanamycin resistance genes that can meet the needs of most users. 


Features

  • Cloning efficiency greater than 90% 

  • IPTG and X-Gal are not required 

  • Accepts a wide range of insert/vector ratios 0.5:1 to 12:1 

  • Accepts insert size from 6 bp to 11 kb

  • The phosphorylation of PCR fragments is not required

  • Accepts blunt end amplicon or DNA fragment (not for sticky ends)

  • Ampicillin and kanamycin selection markers


Storage

-20°C for 24 months

Odoo - Sample 1 for three columns

The plasmid map of pGet II Vector 

Odoo - Sample 2 for three columns

The plasmid cloning sites of pGet II Vector

Contents

Component

Volume

pGet II Vector (25 ng/μl)

23 μl

pGet-For Primer (10 μM)

100 μl

pGet-Rev Primer (10 μM)

100 μl


Primers Sequence                       

pGet-For Primer:               

5'-TCGAAGTTAAAGATGATTACGG-3'

pGet-Rev Primer:

5'-TCTCTCGATAGCATTTCCTGC-3'


Storage

 -20°C for 24 months

 



Ligation Example 1 (NEB T4 DNA Ligase #M0202)

Insert (Blunt end)     X μl (Y ng*)

pGet II (3954 bp)     1 μl (25 ng)

Mix well then add

 

10X T4 DNA Ligase Buffer     2 μl

T4 DNA Ligase                       1 μl

ddH2O                               to 20 μl

Final volume                          20 μl

Mix well then incubate at 16°C or room temperature (20~25°C) for 1 hours.

 

Ligation Example 2 (TOYOBO Ligation High ver2 #LGK-201)

Insert (Blunt end)      X μl (Y ng*)

pGet II (3954 bp)      1 μl (25 ng)

ddH2O                         up to 7 μl

Ligation high ver2              3.5 μl

Final volume                    10.5 μl

Mix well then incubate at 16°C or room temperature (20~25°C) for 5~30 mins.

 

*For 3/1 of Insert/Vector molar ratio:




Transformation

The GetClone™ is compatible with most available competent E. coli cells. Apply 1 ~10 µl of ligation mixture to 10 times volume competent E. coli cells. Perform transformation procedures according to the instruction of the competent cells. Spread the transformed E. coli cells on an LB-ampicillin (50~100 µg/ml) or LB-Kanamycin (50 µg/ml) plate for colony selection.


Recommended colony PCR condition

(SMOBIO’s TP1200 ExcelTaq™ 5X PCR Master Dye Mix is suggested)

Template

Single colony

pGet-For Primer

0.5 µl

pGet-Rev Primer

0.5 µl

5X PCR Master Mix

5 µl

H2O

to 25 µl

Total volume

25 µl

 

Recommended PCR Program

Steps

Temp.

Time

Cycles

Template denature

94°C

2 min

1

Denature

94°C

30 sec

25-40

Annealing

50°C

30 sec

Extension

72°C

30 sec/kb

Final extension

72°C

1 min

1

 


Odoo - Sample 1 for three columns

High Fidelity PCR amplification

Amplification of target gene with HiFi™ DNA polymerase to minimize error rate.

Odoo - Sample 2 for three columns

Gel electrophoresis

Staining amplicons with safe fluorescent dyes, following by observation under blue-light illuminator to minimize damage of DNA amplicons and maximize successful cloning efficiency.   

Safe fluorescent dyes

Blue-light illuminator

Odoo - Sample 3 for three columns

Transformation

Prepare competent cells with high efficiency and transform with time-saving protocol.  

Odoo - Sample 3 for three columns

Colony PCR

Analyze colonies with PCR master mix to save preparation time.