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Saturday, March 2, 2013

Easy Method to Clone a PCR Product


Cloning PCR product or amplicon generated  from non-proofreading (no 3'-5' Exonuclease activity) Taq DNA polymerase is fast method and it has got several advantages.
Bacteria containing plasmids can be frozen, providing a ready supply of amplified material. Because of the variety of available plasmids with different promoters and selectable markers, cloning is also useful when mutations are to be introduced into the fragment before expression, or when sequence tags encoded in the vector are to be added in-frame. The ease with which nucleotide sequences can be added to the ends of PCR products has led to the development of a variety of cloning strategies.

TA Cloning exploits the terminal transferase activity of  DNA polymerases such as Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product.

TA Cloning Plasmid


This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3'-T overhangs. The PCR products with dA overhang, are mixed with this vector in high proportion. The complementary overhangs of "T" vector and PCR product will be ligated under the action of T4 DNA ligase.
By keeping a final extension step in the PCR run can add up A overhangs at the 3' end.

General Process Flow of cloning PCR Product
  1. Set up PCR reaction (Include Final Extension step - 72 dc for 5mins.
  2. Analyse the pcr product on agarose gel.
  3. Gel purify the product (Gel Extraction).
  4. Quantify the gel extracted product (useful for maintaining insert to vector ratio).
  5. Choose 3:1 ratio of insert (varies based on kit).
  6. Ligate the gel extracted PCR product to the vector(Linear plasmid have 5' T overhangs).
  7. Proceed with Transformation into E.coli. (Make sure you have competent cells ready)
  8. Plate 150 ~ 200 uL on LB Agar having suitable antibiotic and other screening agents (X-gal) depending on the vector used.
  9. Incubate the plates overnight at 37dC.
  10. Screen the clones either by colony PCR or by checking the amplification of the insert.
  11. Select Positive clones and proceed with Glycerol Stock Preparation.
  12. Store Glycerol stocks at -80dC(Long term storage) or -20dC.
Various kits are available for cloning PCR product.
Few of them are

InsTA cloning Kit - Thermo Scientific
Qiagen PCR cloning Kit
PCR Cloning Kit -Invitrogen

Notes:
Gel Extraction of the PCR Product will help increase higher yield and higher transformation efficiency.

Reference
CSH Protocols
Internet Sources