Quick10™ Cloning Kit

$385.00

Quick10™ Cloning Kit enables assembly to plate in 10 Minutes!


    6 Reactions - $385.00 (Cat.# 4122 )

Description

Intact Genomics (IG®) propriety Quick10 Cloning kit and DirectPlate® technologies enable simple, rapid and highly efficient cloning. This cloning technology 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.

Quick10 Cloning Kit Process

Key Benefits

  • 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

Applications

  • Streamlined cloning of one or two (<6 kb total) DNA fragments
  • Single PCR product cloning
  • Site directed mutagenesis
  • High throughput cloning

Product Includes
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)

Storage Temperature
-20 ºC for premix, vector and buffer
-80 ºC for Directplate® XL DH10B competent cells

Quick10 Cloning Kit Assembly4122Quick10 Cloning Kit DirectPlate

Please find the video below describing full use of this kit.

4122

Additional information

Reactions

6 Reactions, 50 Reactions, 100 Reactions

Protocol

  1. 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’)
  2. 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.
  3. Dilute the unpurified PCR product 5-10 times utilizing the provided dilution buffer.
    Note: Increasing PCR product purity correlates to increasing cloning efficiency.
  4. Set up the assembly reaction as follows. Insert (s) and vector molar ratio should be 2:1 to produce the highest number of colonies.Quick10 Cloning Kit Assembly Protocol
  5. Mix the reaction mixture thoroughly in 0.2 ml PCR tube.
  6. 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.
  7. 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.
  8. Remove competent cells from the -80 °C freezer and thaw completely on ice and set up the transformation reaction as follows:Quick10 Cloning Kit Trasformation Reaction Set up
  9. 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.
  10. 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
  11. 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.

Quick10™ Cloning Kit Cloning Kit

MSDS - 5x Quick10™-Assemble Premix

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