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Eppendorf Perfectprep Gel Cleanup Kit

Eppendorf Perfectprep Gel Cleanup Kit

Blunt-end Cloning of DNA Isolated Using the Eppendorf Perfectprep Gel Cleanup Kit

George Halley
Eppendorf 5 Prime, Inc.
October 1, 2002 Introduction

Agarose gel electrophoresis is a method used to separate and analyze DNA fragments. This method can be taken a step further; the fragments can be purified from the gel for use in downstream applications. High quality DNA fragments of various sizes (70 bp 10 kb) can be purified from agarose gels using Eppendorfs Perfectprep Gel Cleanup Kit with recovery rates up to 96%. It is important that the isolated DNA is of high quality because most applications are sensitive to inhibitors. Cloning is a common downstream application in which a gel-purified DNA fragment may be used, and successful cloning is a good indication of the purity of the fragment. This application demonstrates the use of gel-purified DNA in a blunt-end ligation.

A 3.49 kb PCR fragment of Lambda DNA was digested with EcoRV to produce a 1.68 kb blunt-ended DNA fragment. The cloning vector, pUC19, was digested with SmaI resulting in a linearized vector with blunt ends. After digesting the DNA and separating it on an agarose gel, the Perfectprep Gel Cleanup Kit was used to purify the fragment and the linearized vector. These were subsequently used as insert and vector in blunt-end cloning reactions which were performed in triplicate. Due to salt sensitivity, blunt-end cloning is more difficult, and this scheme is a stringent test of the cloning capability of gel-purified DNA fragments.

pUC19 contains the lacZ gene allowing for blue/white colony screening as an initial measure of cloning success. White colonies indicate that the lacZ gene was disrupted and the cloning was successful. Based on the number of white colonies produced in this experiment, cloning using DNA purified with the Eppendorf kit was proven to be highly efficient. Subsequent restriction digests on plasmid DNA isolated from white colonies verified that the colonies indeed contained the correctly ligated insert and vector (data not shown). This indicates that DNA obtained from agarose gels using the can be used in sensitive cloning experiments.

Materials

Perfectprep Gel Cleanup Kit (Eppendorf)
Eppendorf PCR Reagents
Taq DNA Polymerase

  • 10x Taq Buffer
  • 25 mM MgCl2
  • 10 mM dNTPs
SeaKem LE Agarose (BioWhittaker Molecular Applications)
1x TBE (Eppendorf)
Ethidium Bromide (Sigma)
Mastercycler gradient (Eppendorf)
Lambda DNA (New England BioLabs)
EcoRV (New England BioLabs)
SmaI (New England BioLabs)
pUC19 DNA (New England BioLabs)
Shrimp Alkaline Phosphatase (Promega)
T4 DNA Ligase (New England BioLabs)
TOP 10 Chemically Competent Cells (Invitrogen)
X-Gal 40 mg/ml in DMF (Sigma)
LB agar plates with 100 mg/ml ampicillin (Teknova)
Molecular Biology Grade Water (Eppendorf)

Methods

PCR1:

A 3.49 kb fragment of Lambda DNA was amplified using the following PCR components. The numbers in parentheses represent the final concentration or amount of th e component in each reaction.

10x Taq DNA Polymerase Buffer (1x)
25 mM MgCl2 (0.5 mM)
10 mM dNTP Mix (0.2 mM)
Taq DNA Polymerase (0.05 U/l)
Template DNA (50 ng)

Primers: 10 M JMF1 5-AAA-AAC-GTC-CGA-GAC-GAA-TG-3 (0.1 M) 10 M JMrev3.6 5-TCA-GCA-TCT-AGC-ATG-CAA-CC-3 (0.1 M) (Integrated DNA Technologies) The Lambda DNA was amplified using an Eppendorf Mastercycler gradient with the following cycling conditions:

94C for 5 minutes

35 Cycles:
94C for 45 seconds
70C for 20 seconds
72C for 2 minutes

72C for 5 minutes
10C forever hold

Restriction Digests:

20 ml of the 3.49 kb PCR product were incubated at 37C for two hours with 20 units of EcoRV in a 30 l reaction. The reaction was stopped by adding 3 l of 10x Gel Loading Buffer and then loaded onto a gel. Several of these reactions were performed in order to obtain enough insert DNA for cloning.

10 g of the vector pUC19 were linearized with 40 units of SmaI in a 40 l reaction for two hours at 25C.

Gel Electrophoresis:

The entire reaction volume (30 l) of each of the EcoRV restriction digests was run on a 100 ml 0.8% SeaKem LE agarose gel containing 2 l of 10 mg/ml ethidium bromide. The gel was run in 1x TBE at 120 V for two hours.

One half of the linearized vector (20 l) was run on a 100 ml 0.8% SeaKem LE agarose gel containing 2 ml of 10 mg/ml ethidium br omide. The gel was run in 1x TBE at 120 V for 1.5 hours.

DNA Purification:

The EcoRV fragments were excised from the gel and purified using the . All samples were eluted in 30 l as described in the manufacturers protocol. DNA concentrations were determined by A260 readings.

The linearized vector DNA band was excised from the gel and purified with the Eppendorf Gel Cleanup Kit and eluted in 30 l. DNA concentration was determined by A260 readings.

Shrimp Alkaline Phosphatase Treatment:

The linearized vector was treated with SAP to prevent re-ligation of the vector ends. 3 l of the vector DNA sample was incubated at 37C for one hour with two units of SAP, 1x SAP reaction buffer and molecular biology grade water in a 30 l reaction, as described in the manufacturers protocol. The enzyme was then inactivated by incubating the reaction at 65C for fifteen minutes.

Ligation:

The ligation reactions were set up based on an insert to vector molar ratio of 3:1. This ratio is based upon the following equation:

(length of insert) (ng of digested plasmid) = ng of insert to use in ligation length of plasmid 1:1 molar ratio = ng insert : ng plasmid
3:1 molar ratio = 3 (ng insert) : 1 (ng plasmid)

Three reactions were performed using insert and vector DNA purified with the Eppendorf Gel Cleanup Kit. 25 l reactions containing 800 cohesive end units (about 6 Weiss units) of T4 DNA ligase and 5% PEG 8000 were incubated at 14C overnight ( 15 hours). Following this, the ligase was inactivated by incubating the samples at 65C for fifteen minutes.

Transformation:

2 l of each ligation reaction were added to 50 l of the TOP 10 competent cells and gently mixed. These cells were kept on ice for thirty minutes and then incubated at 42C for exactly thirty seconds and placed back on ice. 250 l of SOD medium were added to each vial of cells and the vials were then incubated horizontally at 37C for one hour at 225 rpm.

10 l and 30 l of each transformation were plated onto LB agar plates containing 40 l of 40 mg/ml X-Gal and 100 g/ml ampicillin. The plates were incubated at 37C overnight and were checked in the morning for white colonies.

Results

Fig. 1: Blue/white screening of colonies produced from cloning a 1.68 kb blunt-end Lambda DNA fragment into pUC19. 10 l of transformed competent cells were plated. The insert and vector in this ligation were purified from agarose using the . The majority of the colonies on this plate are white, a preliminary indication that the vectors in these colonies contain the insert. Discussion

Insert and vector DNA isolated using the Eppendorf Perfectprep Gel Cleanup Kit can be used in blunt-end cloning, a difficult cloning strategy. In this experiment, all three trials resulted in a high percentage of white colonies suggesting that the majority of the colonies contain the recombinant plasmid. Plasmid DNA from four of these colonies was purified using the Eppendorf Perfectprep Plasmid Min i Kit, and subsequent restriction digests on this DNA confirmed that all four colonies contained the plasmid with the correct insert (data not shown). The DNA purified with the Eppendorf Gel Cleanup Kit performed well in a sensitive cloning procedure and it is assumed that performance will be even better in less difficult cloning such as those involving cohesive ends.

References

  1. Use of the Polymerase Chain Reaction (PCR) process to amplify nucleic acids is covered by U.S. Patent Numbers 4683105 and 4683202 owned by Roche Molecular and requires a license.

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