Tech Tips: Rapid Isolation and Cloning of DNA Inserts, EPICENTRE® Biotechnologies

EPICENTRE Forum 2 (4)

|Tech Tips: Rapid Isolation and Cloning of DNA Inserts|

The use of EPICENTRE's GELase™ Agarose Gel-Digesting Preparation, T4 DNA Ligase, and Ready-Lyse™ Lysozyme Solution can be used to clone DNA fragments - from fragment isolation to analysis of recombinants - in 1.5 days. Table 1 shows the time required for these cloning procedures using Epicentre products.

Table 1. Time Course of EPICENTRE's Cloning Procedures

Procedure

EPICENTRE

Restriction Digest

1-2 hours

Gel Electrophoresis

30 min-1 hour

Fragment Isolation

25 min

Ligation

1 hour

Transformation

overnight

Screening

2 hours

Approximate Total Time

1.5 days

A modified GELase Agarose Gel-Digesting Preparation procedure can be used to purify DNA fragments ready for ligation in 25 minutes.¹ In a representative experiment, one hundred nanograms of an 800 bp EcoRI/Hind III fragment was run on a 1.2% LMP-agarose gel in TAE buffer (Figure 1). The gel was stained with ethidium bromide and the fragment purified using a modified GELase procedure as follows. The fragment was excised from the gel, placed in a 1.5 ml tube and weighed. The agarose plug weighed 100 mg. Two microliters of 50X GELase Buffer were added to the plug, and it was incubated at 70°C for 5 minutes, then at 45°C for 5 minutes. Three units of GELase Preparation were added, and the reaction was incubated for 15 minutes to digest the agarose. (Note: larger agarose plugs require the addition of more GELase Preparation.²)

Figure 1. 800 bp EcoR I/Hind III fragment run on a 1.2% LMP agarose gel before purification using a modified GELase procedure (Lane 2). Lane 1, DNA molecular weight marker.

Ligation Reaction

Following agarose digestion, a ligation reaction was assembled as follows:

10 µl

800 bp fragment in digested agarose

1.5 µl

10X TA buffer (330 mM Tris-acetate, pH 7.8, 660 mM potassium acetate, 100 mM magnesium acetate, 5 mM DTT)

1.5 µl

10 mM ATP

100 ng

EcoR I/Hind III digested pUC19

2 U

T4 DNA Ligase (2 U/µl) (Epicentre)

H₂O to a final volume of 15 µl

The reaction was incubated at room temperature for 1 hour. (Ligation of PCR products into T-vectors should be incubated at 15°C for two hours with 4 U of T4 DNA Ligase.) The reaction was stopped by heating at 70°C for 15 minutes. This step denatures the ligase and improves transformation efficiencies.

Screening of Recombinants

Two microliters of the ligation reaction were transformed into NovaBlue (Novagen) competent cells according to the manufacturer's recommendations and plated on LB ampicillin/tetracycline plates. The plates were incubated at 37°C overnight. Twelve colonies were screened using Ready-Lyse™ Lysozyme Solution in the in-well lysis technique outlined in Table 2.

Table 2. In-well Lysis Screening Protocol.

Pipet 15 µl of protoplast buffer (30 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 25% sucrose, 25 µg/ml RNase A, 8 x 10⁴ U/ml Ready-Lyse™ Lysozyme Solution) into the well of a round-bottom 96-well plate.
Pick colonies with a sterile toothpick and swirl the toothpick in the protoplast buffer for 10-15 seconds.
Patch bacteria from the toothpick onto an agar plate containing the appropriate antibiotic(s). Use a grid so that each colony can be identified.
Incubate the agar plate at 37°C overnight.
Incubate the 96-well plate at room temperature for 10 minutes to allow protoplast formation.
Add 4-8 µl of lysis buffer (0.04 M Tris-Acetate, pH 8.3, 1 mM EDTA, 2% SDS, 5% sucrose, 1 mg/ml bromphenol blue) into the appropriate number of wells on a submerged agarose gel. The amount of lysis solution required depends on the size of the well. The percentage of agarose used should separate plasmids containing inserts from plasmids without inserts. In a separate well on the gel, load a supercoiled kb ladder as a marker.
Add the entire contents of a 96-well plate well of protoplasts to each agarose gel well containing lysis buffer and incubate for 15 minutes to allow the protoplasts to lyse.
Separate the DNA fragments by gel electrophoresis and stain the gel with ethidium bromide.

Three of the randomly screened colonies contained an insert of the correct size (Figure 2). These positive clones are now ready for further analysis. Using this procedure, approximately 40 colonies analyzed on a single gel should yield 10 positive clones.

Figure 2. Results of recombinant screening using the rapid in-well lysis technique. The 800 bp EcoR I/Hind III fragment purified using the modified GELase procedure was ligated with pUC19 and transformed as described in the text. Twelve randomly chosen colonies were screened for the presence of the insert using the described in-well lysis technique (lanes 2-13). Three of the randomly screened colonies contain the correct size insert (lanes 7, 10, 12). Lanes 1, 14, DNA molecular weight markers.

EPICENTRE's GELase Agarose Gel-Digesting Preparation, high activity T4 DNA Ligase and Ready-Lyse Lysozyme Solution can be used to reduce the time required for standard cloning and screening procedures. Using these products, a DNA fragment of interest can be isolated, ligated into a vector and transformed, and the resulting colonies screened in 1.5 days.

1994 EPICENTRE Forum 1 (2), 10.
GELase™ Agarose Gel-Digesting Preparation Product Sheet, Epicentre Technologies.
1995 EPICENTRE Forum 2 (2), 6.