What is the use of blunt end cloning?

The procedure is used for sequencing, building libraries of DNA molecules, expressing coding and non-coding RNA, and many other applications. Blunt-end cloning involves ligating dsDNA into a plasmid where both the insert and linearized plasmid have no overhanging bases at their termini.

How to use strataclone Blunt Cloning buffer?

3 μl StrataClone Blunt Cloning Buffer 2 μl of PCR product (5–50 ng, typically a 1:10 dilution of a robust PCR reaction) or 2 μl of StrataClone Control Insert 1 μl StrataClone Blunt Vector Mix amp/kan 2. Mix gently by repeated pipetting, and then incubate the ligation reaction at room temperature for 5 minutes.

How can I prepare linearized plasmid for blunt-end cloning?

Alternatively, linearized plasmid can be prepared for blunt-end cloning by amplification with a high-fidelity polymerase and subsequent PCR using primers designed with their 5’ ends at the desired insertion site.

What is the maximum length of a blunt PCR product?

The StrataClone blunt PCR cloning kit has been used to clone PCR products up to 9 kb in length. When cloning long PCR products, it is generally advantageous to gel-purify the insert prior to performing the cloning reaction. Long PCR products have been successfully cloned after gel purification using conventional ethidium bromide staining.

Is it possible to clone blunt end DNA fragments with PCR?

Cloning Blunt-Ended DNA Fragments is Hard: pGEM®-T Vectors Can Make It Easier. PCR amplification with a proofreading polymerase, like Pfu DNA polymerase, will leave you with a blunt end.

How do you generate blunt ends in PCR?

Generating Blunt Ends via PCR One important consideration for generating blunt ends by PCR is the presence of 3’ adenines (A) post-amplification. Taq polymerase, which lacks 3’? 5’ proofreading activity, will leave an A on the 3’ end of the amplified product. While this is useful for TA cloning, this will prevent blunt end ligations.