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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Cloning

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Branch: Protocol-Wiki Type: Protocol Intention: For Publication Read/Write Permissions: Open Access

Authors : 01.09.2010 - Christopher Dyer  -  Sci-Mate    24.06.2010 - John Stewart

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Plasmid sub-cloning is a way to place a specific strand of DNA (insert) into a chosen vector.

The protocol presented here, is a very basic and traditional method that works well on 'normal' plasmids around 5 kb, with inserts between 700 and 2 kb.

In my humble opinion, while this is an effective and trusted method in the hands of experienced molecular biologists, considerable time, effort and money can be saved using modern methods, such as Gateway Cloning.

Contents 1 Overview 2 Restriction Enzyme Digest 3 Electrophoresis Gel Separation and DNA Purification 4 DNA Ligation 5 Bacterial Transformation

[edit] Overview

1. Digest both insert and vector with complimentary restriction enzymes.
   
2. Separate both insert and vector strands of DNA (based on size) via an electrophoresis gel.
   
3. Cut and purify insert and vector DNA.
   
4. DNA Ligation.
   
5. Transform bacteria with ligated (sub-clone) DNA, which can be selectively grown on antibiotic plates if the vector contains a gene confering resistance.
   
6. Mini-prep plasmid DNA from isolated cultures to identify successful sub-clones by restriction enzyme digest.
   

[edit] Restriction Enzyme Digest

The choice of enzymes that will ideally create two different sticky ends on the insert, that will bind to complimentary sticky ends created on the vector is the basis of the 'cloning strategy'.

It is recommended to digest a relatively large amount of insert DNA, compared to vector. At least 1ug.

For example:

1 ul Plasmid DNA in TE [minimum 1ug/ml]

1-2 ul of Buffer (eg, FOPA buffer)

1ul REDigest

Distilled water to 10ul

[edit] Electrophoresis Gel Separation and DNA Purification

Both digests of DNA should be loaded into separate lanes of agarose gel containing ethidium bromide (1ug per lane- multiple lanes if necessary).

After running marker to the end of the gel, both insert and vector should be identified under a 'gentle' UV, and cut out with a razor blade or scalple.

DNA can then be purified using a commercial kit or via silica purification.

[edit] DNA Ligation

1ul DNA Ligase
1ul DNA Ligase Buffer (10X)
1ul Vector [1ug/ml]
7ul Insert [1ug/ml]

AND, a vector only control (as above, but without insert (use distilled water or TE instead))

1. Mix well; leave >3hrs at room temperature.
   

If the sub-cloning does not work effectively, one possible solution is to titrate various concentrations of vector : insert. For example starting at 1:1; 1:2; 1:4; 1:8; 1:16

[edit] Bacterial Transformation

1. Prewarm agar plates (37⁰C) for >1hr.
   
2. Defrost 100ul CaCl competant cells (on ice); leave 5’ – 20’ on ice;
   
3. Spread cells onto warm plates and incubate o/n at 37⁰C.
   NB// Include Vector only control with H20.