Quick10™ Cloning Kit enables assembly to plate in 10 Minutes!
Quick10™ Cloning Kit
Intact Genomics (IG®) propriety Quick10™ Cloning kit and DirectPlate® technologies enable simple, rapid and highly efficient cloning. The Quick10™ Cloning kit utilizes homologous assembly which allows for nearly 100% cloning accuracy. This kit enables the direct cloning of one or two DNA fragment(s) (<6kb total) through PCR into linearized cloning-ready Ig® AmpR/KanR-pCR vector (4 kb, provided in this kit). The PCR fragments can be generated by Intact Genomics high fidelity i7® DNA polymerase or other high fidelity DNA polymerases. Primers need to have 12 to 15 bases of homology at Ig® AmpR/KanR-pCR vector linear ends where the DNA fragment(s) or PCR product(s) needs to fuse. The unpurified PCR product (s) is then diluted 5-10 times by the dilution buffer included in this kit. No purification step is required. The diluted PCR product (s) and linearized Ig® AmpR/KanR-pCR vector can then be assembled in just 7 minutes at 37 ºC, followed by a 3 minutes transformation step on ice utilizing our Directplate® XL DH10B competent cells (included in this kit). DirectPlate® cells eliminate heat shock, lengthy incubations, and time-consuming outgrowth procedures. Utilizing this kit, DNA/PCR product(s) can be assembled/transformed in 10 minutes vs. other seamless/fusion cloning, conventional T/A, or restriction ligation cloning and transformation methods that can take up to 2 hours or more.
- Combination of high fidelity PCR, assembly, and DirectPlate® transformation cloning technologies.
- Combination of homologous assembly and ccdB selection (vs. T/A or restriction ligation methods) displays <1% false-positive clones or nearly 100% cloning accuracy.
- Assembled/transformed in 10 minutes. Less time and less effort spent on cloning, transformation, and positive clone screening/identification.
- Single selection vector or custom vector available upon request
- Streamlined cloning of one or two (<6 kb total) DNA fragments
- Single PCR product cloning
- Site directed mutagenesis
- High throughput cloning
1) 5x Quick10-Assemble™ Premix
2) Ig® AmpR/KanR-pCR vector – cloning ready
3) Dilution buffer
4) Directplate® XL DH10B competent cells (6×50 µl)
-20 ºC for premix, vector and buffer
-80 ºC for Directplate® XL DH10B competent cells
Please find the video below describing full use of this kit.
6 Reactions, 50 Reactions, 100 Reactions
- Design PCR primers for the DNA of interest with 12 to 15 bp at 5’-extensions to the ends of the linearized Ig® AmpR/KanR-pCR vector sequence (e.g. 5’-extension forward primer: 15bp homology 5’-GCGAATTCTGCAGAT-3’ and 3’-extensions reverse primer: 15bp-homology complementary strand 5’-TAGATGCATGCTCGA-3’)
- Amplify the DNA of interest with Intact Genomics i7® High-Fidelity DNA Polymerase 2x Master mix (cat. #3257 ) or any other high fidelity DNA polymerase. Run the PCR product on an agarose gel to determine the integrity of the PCR product.
- Dilute the unpurified PCR product 5-10 times utilizing the provided dilution buffer.
Note: Increasing PCR product purity correlates to increasing cloning efficiency.
- Set up the assembly reaction as follows. Insert (s) and vector molar ratio should be 2:1 to produce the highest number of colonies.
- Mix the reaction mixture thoroughly in 0.2 ml PCR tube.
- Incubate the reaction mixture at 37 °C for 7-15 minutes, then place on ice.
Caution: The assembly reaction maximum incubation is 30 minutes. Longer incubation times decrease cloning efficiency.
- Use 1.0 to 5.0 µl of the reaction mixture and transform into DirectPlate® XL DH10B competent cells (included in this kit).
Note: DirectPlate® competent cells are optimized for high transformation efficiency and save significant time.
- Remove competent cells from the -80 °C freezer and thaw completely on ice and set up the transformation reaction as follows:
- Mix by gently pipetting up and down a few times then place on ice or room temperature for 3-10 minutes. Caution: Longer incubation times decrease cloning efficiency.
- Spread 25 to 50 μl from each transformation directly onto LB plate containing 100 μg/ml ampicillin. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. Use a sterilized spreader or autoclaved ColiRoller™ plating beads to spread evenly.
Caution: Drying the plate too much will decrease cloning efficiency
- Incubate the plates overnight at 37 °C.Note: The procedures above are for Ig® AmpR/KanR-pCR vector containing Ampicillin and Kanamycin resistant markers. To prevent self ligation, Ig® AmpR/KanR-pCR has the ccdB gene.