Lab Report- 1
Name of Experiment: Isolation of Plasmid DNA by Alkaline Lysis
Method and Gel Electrophoresis
Submitted To
Dr. Suraia Nusrin (DSN)
Chairperson, Associate Professor
Department of Genetic Engineering & Biotechnology
East West University
Course Code: GEB 402
Course Title: Advances in Recombinant Gene Technology
Section: 1
Submitted By
Samia Binte Bashar
ID: 2020-1-77-048
Date of Submission: 12th March, 2025
Experiment name: Isolation of plasmid DNA by alkaline lysis method and Gel
Electrophoresis
Principle:
Most plasmid isolation methods rely on the notable molecular weight differential
between plasmid and chromosomal DNA. The alkaline lysis technique uses NaOH
to lyse cells, and KAc generates a precipitate with high molecular weight
chromosomal DNA.While alkaline pH denatures both genomic DNA and proteins.
Because the solution is neutralized, covalently closed plasmid DNA reanneals
quickly due to the connectivity of both single-stranded DNA rings. Lowering the
temperature in the following phase of the method causes the plasmid to remain in
the supernatant, which can then be precipitated using isopropanol. The pGLO
plasmid will be isolated from E. coli DH5 alpha using the alkaline lysis technique.
Solution and Reagents:
For Plasmid Isolation
Solution 1 Solution 2 Solution 3
50 mM Glucose 0.2 N NaOH 5 mM Potassium acetate
(Filter Sterilized) (Freshly Diluted from 10 60 ml
N Stock)
25 mM Tris-HCL, pH 8.0 1% SDS Glacial acetic acid 11.5
ml
10 mM EDTA, pH 8.0 H2O 28.6 ml
Autoclave for 15 min for 100 ml solution 3 at 10 lb/sq in on liquid cycle, and store
at 4 degree C.
For Gel Electrophoresis
1. Electrophoresis grade agarose
2. TAE Buffer
3. Gel Loading Buffer
4. Ethidium Bromide
5. Plasmid DNA samples
6. Horizontal gel electrophoresis equipment+ power pack
Procedure:
For Plasmid Isolation
1. Transferred a bacterial colony into 100 m of LB containing appropriate
antibiotics.
2. Incubated the culture overnight at 37°C.
3. Poured 1.5 ml of the bacterial culture into the eppendorf tube.
4. Centrifuged the culture at 3,500-4,000 pm for 2 min.
5. Removed supernatant and dried the pellet.
6. Added 200 μl of solution 1 (ice cool) and resuspended bactorial cells by
vigorous shaking.
7. Added 400 μl of solution 2 at each tube, mixed well but did not vortex.
8. Stored in ice for 5 min. strictly.
9. Added 300 μl of solution 3 and shook upside down.
10.Stored the tube on ice for 5-7 min.
11.Centrifuged at 13,000 pm for 10 min.
12.Transferred the supernatant in a fresh eppendorf tube (700-750pl).
13.Added equal volume of phenol: chloroform: IAA (25:24:1).
14.Centrifuged at 13,000 rpm for 10 min.
15.Transferred the supernatant in a phase into another eppendorf tube.
16.Precipitated the DNA with equal volume of ice cold isopropanol.
17.Shook and allowed the mixture to stand for 2 min at room temperature.
18.Centrifuged at 13,000 pm for 10 min.
19.Removed supernatant and dried the pellete.
20.Rinsed the pellete with 1 ml of ice cold 70% ethanol.
21.Centrifuged at 13,000 rpm for 10 min.
22.Removed the supernatant and dried the pellet.
23.Added 20 μl TE solution in each eppendorf tube and then collected all plasmid
DNA in one eppendorf tube.
24. Run 5-7 μl of plasmid with 2 ml DNA loading dye on an 0.8% agarose gel,
with DNA markers.
25. Found out the approximate molecular weight of your plasmid by comparing it
with known DNA markers.
For Gel Electrophoresis
1. Prepared a 1.0% agarose gel as demonstrated by class demonstrator.
2. Loaded the samples in the agarose gel in the slots present.
3. Commenced electrophoresis (70 V) using TAE buffer.
4. Stained the gel with ethidium bromide by soaking for 30 minutes.
5. Observed the DNA bands by planning the gel on a UV trans illuminator.
Result:
Figure :Gel electrophoresis of pGLO plasmid from E coli.
The plasmid DNA samples were loaded onto the gel, with a 1 kb DNA ladder
serving as a size reference. The ladder displayed distinct bands ranging from
10,000 bp to 300 bp. However, the sample lanes exhibited smearing instead of
well-defined bands, indicating low DNA yield and potential degradation. While
some bands appeared around 1,500 bp, their exact size could not be determined
due to the smearing.
Discussion :
The result of the electrophoresis should give 5400 bp but in our experiment we got
1500 bp. The smearing and lack of clear bands in the gel suggest several potential
issues during the experiment. Improper well loading could have led to sample
leakage or uneven distribution, affecting band resolution. Errors in the separation
of the supernatant during the alkaline lysis method may have resulted in
contamination with genomic DNA or cellular debris, reducing plasmid purity.
Additionally, reagent inconsistencies, such as incomplete mixing of lysis or
neutralization buffers, could have impacted DNA precipitation and integrity. The
presence of residual proteins or RNA may have further interfered with
electrophoresis, causing smearing. To improve results, careful handling during
each step, ensuring proper buffer preparation, and incorporating purification steps
like RNase treatment and ethanol precipitation could enhance DNA quality and
yield.
Precaution :
● Appropriate personal protective equipment, including a lab suit, gloves, and
eye protection, should be worn.
● Phenol and chloroform should be handled with caution.
● As chloroform is heavy, it should be transferred quickly during the
experiment to minimize loss.
● The same pipette tip should not be used for sampling different reagent
mixtures.
● Thorough and careful handling of the pellet should be ensured when adding
solutions or reagents or when resuspending it.
● Care should be taken to avoid pipetting any part of the pellet when removing
the supernatant from the top of the micro tube .
● When mixing chemicals, the formation of bubbles inside the micro tube
should be avoided, as this can interfere with the procedure.
