Vector construction technology is one of the commonly used technologies in molecular biology and genetic engineering research. When using traditional methods to construct complex vectors with multi-segment ligation, it is usually necessary to construct multiple intermediate vectors, and multiple ligation and transformation are time-consuming and labor-intensive. As a new cloning technology, homologous recombination cloning can insert one or more target fragments of any site of the plasmid, and the construction of the vector can be completed with only one step of recombination, which greatly improves the work efficiency.
1. What is the principle of homologous recombination cloning?
2. Why are YEASEN's products excellent?
3. Other Cloning Related Products from YEASEN
1. What is the principle of homologous recombination cloning?
After the homology arms are introduced into both ends of the target fragment, the 5'-end exonuclease activity of the recombinase degrades the linearized vector and the 5'-end of the target fragment DNA, and the resulting single-stranded ends are paired; Then, the temperature rises gradually, the DNA polymerase activity of the recombinase replenish the missing bases; finally, the ligation activity of the recombinase repairs the gap between the bases, then we obtain a complete double-stranded DNA.
Figure 1. Schematic diagram of the principle of homologous recombination
2. Why are YEASEN's products excellent?
The homologous recombination cloning kit developed by YEASEN has been widely praised since its relase. Good reputation comes from feedback from the public. Good reputation comes from feedback from the public. Next, Xiaoyi will take you to see the feedback from our customers!
Hieff Clone™ Plus One Step Cloning Kit (Cat# 10911)
Feedback 1: Shandong Normal University - 1300 bp single fragment cloning
Figure 2. Single fragment cloning of 1300 bp was performed with the Hieff Clone™ Plus One Step Cloning Kit. A: Electrophoresis of the inserted target DNA; B: Experimental group clone growth status; C: Positive clones detected by colony PCR (the size of the bands are all correct).
Feedback 2: Zhejiang University - 400 bp single fragment cloning
Figure 3. Single fragment cloning of 400 bp was performed with the Hieff Clone™ Plus One Step Cloning Kit. A: Electrophoresis of linearized vector plasmid (Marker 8000); B: Electrophoresis of the inserted target DNA (Marker 5000); C: Growth status of clones in experimental group; D: Positive clones detected by colony PCR (the size of the bands are all correct).
Feedback 3: Sichuan University - 2445 bp single fragment cloning
Figure 4. Single fragment cloning of 2400 bp was performed with the Hieff Clone™ Plus One Step Cloning Kit. A: Electrophoresis of pFastBac I plasmid (with signal peptide); B: Electrophoresis of pFastBac I plasmid (with signal peptide) digested by EcoR I and Hind III; C: Growth status of clones in experimental group; D: Positive clones detected by colony PCR (the size of the bands are all correct).
Feedback 4: Tianjin University of Traditional Chinese Medicine - 2290 bp single fragment cloning
Figure 5. Single fragment cloning of 2290 bp was performed with the Hieff Clone™ Plus One Step Cloning Kit. A: line 1 is the linear vector, and line 2 is the target DNA fragment; B: Growth status of clones in experimental group; C: Positive clones detected by colony PCR.
Hieff Clone™ Universal One Step Cloning Kit (Cat# 10922)
Feedback 1: Institute of Botany, The Chinese Academy of Sciences - 975 bp single fragment cloning
Figure 6. Single fragment cloning of 975 bp was performed with the Hieff Clone™ Universal One Step Cloning Kit. A: Electrophoresis of linearized vector; B: Electrophoresis of the inserted target DNA; C: The upper picture shows the growth status of clones in the experimental group, and the lower picture shows the positive control group; D: Positive clones detected by colony PCR.
Feedback 2: Wuhan University - 1600 bp+1700 bp two- fragments cloning
Figure 7. Two-fragments cloning of 1600 bp + 1700 bp were performed with the Hieff Clone™ Universal One Step Cloning Kit. A: Electrophoresis of the inserted target DNA, lane 1 is the 1.6 Kb target DNA, lane 2 is the 1.7 Kb target DNA; B: Electrophoresis of linearized vector; C: Growth status of clones in experimental group; D: Positive clones detected by colony PCR.
Feedback 3: Tsinghua University - 3600 bp single fragment cloning
Figure 8. Single fragment cloning of 3600 bp was performed with the Hieff Clone™ Universal One Step Cloning Kit. A: Electrophoresis of the inserted target DNA; B: Control is the transformation status of the control group in the product, BD is the growth status of clones in experimental group; C: Positive clones detected by colony PCR.
Feedback 4: Nanjing Agricultural University-two fragments and four fragments recombination cloning
Figure 9. Two- and four-fragment cloning were performed with the Hieff Clone ™ Universal One Step Cloning Kit. A: Lane. 1-9 are PCR products, lane 6 is the linearized pACYC177, lane 7 is the linearized pBR322; B: Electrophoresis of the inserted target DNA; C: The picture shows the colony growth after 12 hours of plating, the picture with “×”is to verify the activity of DH5α, pA2 represents the transformation of the two-fragment recombinant plasmid, pA4 represents the transformation of the four-fragment recombinant plasmid, and the same is true for pB2 and pB4. D: Positive clones detected by colony PCR (pA using primers of fragments No. 2 and 5, pB using primers of fragments No. 4 and 5) and identified by restriction enzyme digestion. The identification results of both methods were correct.
3. Other Cloning Related Products from YEASEN
YEASEN has a complete line of molecular cloning products, including PCR, cloning and transformation. In the table below, we list all molecular cloning related reagents and their applications from YEASEN, you can choose the most suitable reagent for your experiment.
Table 1. Other Cloning Related Products from YEASEN
Product orientation |
Product Name |
Cat# |
Specifications |
One-step cloning with single fragment |
Hieff Clone™ Plus One Step Cloning Kit (inquire) |
10911ES20 |
20T |
One-step cloning with 1-5 fragments |
Hieff Clone™ Plus Multi One Step Cloning Kit (inquire) |
10912ES10 |
10T |
One-step cloning with 1-6 fragments |
10922ES20 |
20T |
|
Normal PCR amplification |
10102ES03 |
1mL |
|
Normal PCR amplification |
2×Hieff™ Ultra-Rapid HotStart PCR Master Mix (with Dye)
|
10157ES03 |
1mL |
Complex DNA template amplification |
10154ES03 |
1 mL |
|
Nucleic acid extraction by column |
MolPure™ Plasmid Mini Kit (inquire)
|
19001ES50 |
50T |
Nucleic acid extraction by column |
MolPure™ Endo-free Plasmid Mini Kit (inquire) |
19021ES50 |
50T |
Nucleic acid extraction by column |
MolPure™ Endo-free Plasmid Maxi Kit(inquire) |
19036ES10 |
10T |
Nucleic acid extraction by column |
MolPure™ Gel Extraction Kit (inquire) |
19101ES50 |
50T |
Nucleic acid extraction by column |
MolPure™ PCR Purification Kit PCR (inquire) |
19106ES50 |
50T |