Molecular Cloning - 4. PROTOCOL(보류)

4. PROTOCOL

4.1. Preparation

Ensure that good-quality DNA preps are available for the receiving vector and any vectors used to retrieve the insert (see Isolation of plasmid DNA from bacteria).

 

 

Identify the restriction enzymes needed and their buffer requirements for single and double digests.

For PCR product inserts:

Add the sequence of any desired restriction site(s) to the 50 end of each primer (see Explanatory chapter: PCR -Primer design).

 

It is a good idea to precede these sites with a few random nucleotides, such as GATC, since some restriction enzymes can cut only at sites surrounded by doublestranded DNA. Note that these regions should be excluded from annealing temperature calculations since they do not contain any homology to the target sequence (Fig. 7.2).

 

Once you obtain the primers, run the PCR reaction and gel-purify the product, eluting in 30 ml of ddH2O. Design one or more strategies to test for vectors that carry the correct insert (in the appropriate orientation).

Examples:

1. Identify restriction enzymes that cut inside (and outside) of the new insert, producing distinct bands.

2. Design PCR primers, one outside and one inside the insert, to use for colony PCR.

3. Identify or design primers that can be used to sequence all or part of the insert.

 

 

4.2. Duration

 

5. STEP 1 RESTRICTION DIGESTS OF VECTOR AND INSERT

5.1. Overview

Linearize the receiving vector by restriction digest and cut out the insert from another vector or digest a gel-purified PCR product to create sticky ends.

 

Linearize 선으로 만들다

 insert  끼우다

 

 

If using DNA oligos, anneal the two single-stranded pieces to form a double-stranded insert with sticky ends according to the manufacturer’s directions and go directly to Step 2

 

 

 

5.2. Duration

2.5–3 h

1.1 Set up the restriction digests for both the vector and the insert in 1.5-ml microcentrifuge tubes:

 

1.2 Incubate at 37 C for 1 h.

1.3 Pour a 0.8% agarose gel in 1*TAE buffer containing ethidium bromide (see Agarose Gel Electrophoresis).

1.4 Add 10 ml of 6 DNA gel loading dye and load onto the agarose gel. Run the gel at 100 V for 30–45 min

1.5 Gel-purify the receiving vector and the insert, using a DNA Gel Purification kit. This purification step is essential as it removes any enzymatic activity that might interfere with the ligation reaction. When running the gel, include an uncut vector control to confirm that the digests are complete. Any residual uncut plasmid will produce a high background of negative colonies

1.6 Optional: To create blunt ends, fill in any overhangs left by restriction enzymes. Set up the following reaction in a 1.5-ml microcentrifuge tube:

1.7 Incubate at room temperature for 30 min.

1.8 Stop the reaction with 1 ml of 0.5 M EDTA.

1.9 Purify using a PCR purification kit or by gel-extraction.

 

5.3. Tip