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