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Learn how to use DNA ligase - a most powerful tool
used in DNA cloning
techniques.
Understand cloning techniques which are used in
genetic engineering
Following DNA ligation, understand the techniques
of bacterial transformation.
Understand how bacteria propagate in presence of
antibiotic.
Learn the concept of the protein expression and
use of X-gal and IPTG.
Understand identifiable DNA traits which make white
or blue colonies.
Learn how to make antibiotic resistant growth media.
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DNA cloning is a very common technique
used in molecular biology to study gene structure,
function and expression. T4 DNA Ligase is used to
ligate fragments of DNA with plasmid DNA, resulting
in the formation of recombinant closed circular
plasmid DNA. Resulting circular DNA is transformed
in E.coli cells by using antibiotic resistant agar
plates. Bacterial colonies on agar plates are used
to isolate (plasmid preparation) DNA of interest.
The experiment includes, a) precut plasmid DNA,
b) precut fragment with matching overhangs, and
c) T4 DNA ligase enzyme. The efficiency of ligation
and transformation could be assayed by counting
the number of colonies on bacterial plates. |
Time Needed
to Complete the Experiment:
Grow E.coli cells (16 hours) or use frozen competent cells.
Preparation of small scale competent cells: 20 minutes.
Preparation of antibiotic resistant plates: About 40 minutes.
Plates containing agar media can
be stored in refrigerator for up to three months.
Transformation of plasmid DNA to competent cells: 30 minutes.
Inoculation of plates with
transformed E.coli cells: 5 minutes.
Results (after 16-24 hours of incubation of plates at
37 °C or about two days of incubation at
room temperature.
Staining time:
2-3 hrs for methylene blue stain or instant results
with ethidium bromide stain.
Kit includes following
items:
Uncut plasmid DNA (as control). Blue and white colony
making plasmids.
Pre-cut plasmid DNA.
Pre-cut DNA fragment with matching overhangs.
DNA Ligase enzyme.
Ligase buffer.
E.coli cells.
LB Media for bacterial growth.
Agar Media for plates.
Petri plates.
X-Gal/IPTG Solution.
Transformation buffer .
Double autoclaved sterile water.
Antibiotic (Ampicillin)- enough for making plates
Microfuge tubes.
Sterile inoculating loops.
Instructions sheet.
Blank sample for practice on agarose gel.
Other Requirements:
Microwave, Incubator (optional), Glassware, Hot Gloves
| Catalog# |
Description |
Price(1
Kit) |
Price(3
Kits) |
Price(5 Kits)
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| 313-1 |
Cloning (ligation) and bacterial transformation.
30 preps |
$89 |
$256 |
$409 |
| *Extended
kit includes extra reagents (for visualization of
DNA during electrophoresis) such as loading pipet,
agarose, microfuge tube rack, gel buffer, standard
DNA, staining solution and agarose electrophoresis
videotape etc. Note: Due to restriction enzymes,
cold shipment charges will apply. |
|
Enough for 30 plates (10 each of white, blue and red colonies)
