Hetero-Stagger PCR Cloning Kit, DynaExpress

Product#: DS158
$265.00
Availability:
Ships in 1-2 weeks

Hetero-Stagger PCR Cloning Kit, DynaExpress

Cat. No. DS158
Size 1 Kit
Store -20oC
Storage Conditions

Box 1 should be stored at -20℃. Box 2 should be stored at -80℃ directly from a dry ice shipping box.
Bottle should be stored at room temperature (or -80℃)
Competency of JM109 > 1 × 108 cfu/μg?50μl cell (pBR322)
Genotype of E coli strain JM109  recA1, supE44, endA1, hsdR17, gyrA96, relA1, thi, Δ(lac-proAB), F′[traD36, proAB+, laqIqZΔM15] 

Description
Hetero-Stagger PCR Cloning Kit enables highly efficient and fast cloning of PCR products. This novel method does not require any enzymatic procedures. pHST Vector is included.

TA cloning of PCR product is known as a much less effi cient cloning method than cohesive end and blunt end cloning method.
DynaExpress Hetero-Stagger PCR Cloning Kit enables highly effi cient and fast cloning of PCR products. This novel method does
not require any enzymatic procedures such as restriction enzyme, ligase, exonuclease, uracil DNA glycosylase and Cre-loxP
recombinase reactions.
DS158_fig1.jpg
Instead, the method requires 2 PCR reactions. The PCR reactions are set up to generate 2 PCR products containing diff erent
extra terminal sequences. The products and a compatible vector harboring 9 bases single-strand extensions with complementary
sequences, pHST, are mixed, heat-denatured and annealed to form a heteroduplex product. Ligation reaction is not required,
because the complementary extension of the vector and the insert are so long. The mixture of annealed vector and the PCR
products can be used directly for the transformation of chemical competent cells.

Features
  • High ligation efficiency.
  • Simple procedure (mix, heat and anneal).
  • Does not require any enzymatic reactions.
  • Direct use for transformation after annealing.
  • Choice of insert orientation.
  • Applicable to both proofreading and non-proofreading DNA polymerase.
  • Total time from PCR product to plating is just one and a half hours.
Example Data
Several amounts of about 1 kb PCR fragments were cloned according to the standard protocol using the DynaExpress Hetero-Stagger PCR Cloning Kit. Half amounts of the transformed competent cells (150 μl) were spread onto LB agar plates. The white bars and the blue bars show the numbers of white colonies and blue colonies, respectively. There are few blue colonies!

DNA Ligation Kit ver.2, DynaExpress DNA Ligation Kit ver.2, DynaExpress
DNA Ligation Kit ver.2 enables highly effi cient ligation of cohesive or blunt end DNA fragments within 5-30 minutes at 16℃ -25℃ . Simple ligation reaction can be started by adding 2 × Ligation Buff er and Ligase Mixture to a mixture of vector and insert DNA solution. The ligation reaction mixture can be used directly to the transformation of chemically competent cells.
  • High ligation effi ciency.
  • Simple and quick procedure.
  • Use ligation mixture for transformation directly after ligation reaction.
Transformation protocol 
  1. Thaw the competent cells on ice (50 µl in a tube of each transformation).
  2. Add 5 µl of the annealing mixture directly into the competent cells and mix by flicking gently.
  3. Incubate the tube on ice for 20 minutes.
  4. Heat Shock the cell by placing the tube in a 42°C water bath for 45 seconds. Do not mix or shake.
  5. Remove tube from the 42°C bath and place it on ice for 2 min.
  6. Transfer the cell to 15 ml sterilized culture tubes containing 250 µl of SOC medium (pre-warmed from room temperature to 37°C). Culture the cell at 37°C for 1 hr in a shaker. 
Annealing Protocol 
  1. Perform the two separate PCR reactions to produce two PCR products.
  2. Set up the 15 µl annealing mixture on ice as follow:
    • PCR product 1 and PCR product 2 X µl
    • Annealing buffer 8 µl
    • pHST1 vector (15ng/µl) 2 µl
    • Distilled water variable
    • Total volume 15 µl
  3. Heat to 95℃ for 5 min and cool gradually to room temperature for 5 min.
  4. Transform chemically competent E.coli cells JM109 with 5 µl of annealing mixture
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