Practical #2: Extraction of genomic DNA from E.Coli

Practical #3: Agarose Gel Electrophoresis

*CELL BIOLOGY PRACTICAL

Bertrand OngChan JianPengSalanne Lee

*OVERVIEW

*Introduction

*Materials & Procedure

*Results & Discussion

*Conclusion

*Q&A

*INTRODUCTION: DNA

*DNA (Deoxyribonucleic acid)

*Contains genetic makeup of all living organisms

*Bases: Adenine (A), Thymine (T), Guanine (G), Cytosine (C)

*Base + Phosphate Group + Deoxyribose Sugar = Nucleotide

Nucleotide!

*GENOMIC DNA OF E.COLI

*Escherichia Coli, or E.Coli are prokaryotic cells (no nucleus)

*Circular DNA, also known as PLASMIDS found in nucleoid

*Many strains of E. Coli exists

*OBJECTIVES OF EXPERIMENT

*Extract genomic DNA from bacterial cultures

*Determine the yield and quality of DNA using Nanodrop

*Separate genomic DNA using agarose gel electrophoresis

*Learn how to capture a gel image using a UV transilluminator

*INTRODUCTION: THEORY

*Nucleic acid quantification is usually used to determine the concentration of DNA and RNA in a mixture.

*Nanodrop (spectrophotometer) expose a sample to ultraviolet light

* photo-detector measures the light that passes through the sample

* The more light absorbed by the sample, the higher the nucleic acid concentration.

* Agarose gel electrophoresis is a frequently used technique in molecular biology to separate DNA or RNA fragments by size.

* determine the size of PCR products and to isolate DNA fragments after restriction enzyme digestion

PR

AC

TIC

AL

#2

PR

AC

TIC

AL

#3

*MATERIALS & EQUIPMENT

*E.coli bacterial cultures

*Qiagen DNeasy blood and tissue kit

*Microcentrifuge tube

*Microcentrifuge

*Agarose gel powder

*Conical flask

*Gel red, DNA ladder, loading dye, TBE buffer

*Gel tanks

*Power pack

*Extracted E.coli DNA

*Bio-Rad Doc imaging system

*PROCEDURE: DNA Extraction of E.Coli

1. The bacterial cells are spun down for 10 min at 4000 rpm.

2. Pellet is suspended in 180µl Buffer ATL. Cell suspension is then pipetted into a microcentrifuge tube.

3. 200µl Buffer AL is then added to the sample, mixed and incubated at 56⁰C for 10min.

4. 200µl ethanol (96 - 100%) is then added to the sample and thoroughly mixed.

*PROCEDURE: DNA Extraction of E.Coli

5. The mixture is pipetted into the DNeasy Mini spin column and centrifuged at 8000 rpm for 1 min. The filtrate is discarded.

6. The DNeasy Mini spin column is placed back into the collection tube. 500µl Buffer AW1 is added and centrifuged at 8000 rpm for 1 min. The filtrate is discarded.

7.Step 6 is repeated but this time with Buffer AW2 and centrifuging of the mixture occurs at 14000 rpm.

8. The DNeasy Mini Spin column is then placed into a microcentrifuge tube and 200µl Buffer AE is pipetted directly onto the DNeasy membrane. The mixture is incubated at room temperature for 1 min and centrifuged for 1 min at 8000 rpm to elute.

1. 1.2g of agarose is weighed.

*PROCEDURE: AGAROSE GEL ELECTROPHORESIS

2. 100ml of 1X Tris Borate-EDTA (TBE) buffer is added.

4. 5µl of gel red is added into the molten agarose

5. It is then poured into a gel caster (pre-prepared with a well comb.

*PROCEDURE: AGAROSE GEL ELECTROPHORESIS

6. The well comb is gently removed from the gel once the gel has set.

7. C1X TBE buffer is then poured into the gel tank until the gel is submerged

8. 2µl of loading dye is then mixed with 10µl of extracted DNA on parafilm and loaded into the wells. 5µl of 1 kbp DNA ladder is loaded into 1 of the wells

9. Electrophoresis is then run at 90V for 40 min.

*PROCEDURE: AGAROSE GEL ELECTROPHORESIS

10. The DNA bands are then visualised using the UV transilluminator (Bio-Rad Gel Doc imaging system)

*“Nanodrop” Result

We have collected our results

The following value is the ratio of A260/A280 ratio change

• Value:1.90 (A260/A280)

• Value: 0.99 (A260/A230)

• DNA sample PURE ! (within range of 1.8)

• Nucleic Acid

Contain Contaminants (out of range)

*UV transilluminator

UV transilluminator with an image analysis software

• Makes use of UV light to illuminate the resultant DNA fragments

• Prior to Illumination, fluorescent dye is added to the gel.

e.g. Ethidium bromide, gel red

*Visualizing Image

• Consist of illuminated DNA fragments (In white)

• With white markings of different lengths

• Usually includes a DNA Marker

Our Result

* Visualizing Image (The Principle)

*The further the ‘white pattern’ travels, the smaller the DNA molecule.

*Allows differentiating of the molecular mass of DNA molecules

*Conclusion

(Summary)

*Genomic DNA was extracted in the 2nd practical and the quality of the DNA was experimentally determined to be pure (except for the purity of nucleic acids).

*In the 3rd practical, agarose gel electrophoresis was conducted and the visualizing image was collected.

P.S. PLEASE BE NICE

*Q&A

*THANK YOU~!

*REFERENCES

1. Microcentrifuge tube [online]. USA: Sorenson, BioScience, Inc. Availble from: http://www.sorbio.com/content/65-mct-nat-s_1.jpg[Accessed 6 July 2011]

2. Nucleotide [online]. USA: Sciencey Splurge. Availble from: http://scienceysplurge.files.wordpress.com/2010/10/nucleotide.jpg [Accessed 6 July 2011]

3. E.Coli [online]. USA: Wordpress. Available from: http://2012patriot.files.wordpress.com/2011/06/ecoli2.jpg [Accessed 6 July 2011]

4.E.Coli: Black & White [online]. USA: Wikipedia. Available from: http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/EscherichiaColi_NIAID.jpg/250px-EscherichiaColi_NIAID.jpg [Accessed 11 July 2011]

5. DNA Electrophoresis Size Standards [online]. USA: Bio-Rad. Available from: http://www.bio-rad.com/webroot/web/images/lsr/products/imaging_bioinformatics/product_overlay_content/global/lsr_geldoc_sizestds.gif [Accessed 11 July 2011]