Isolation And Quantification Of Plasmid DNA from Bacteria

Question 1

What are the steps of the plasmid mini prep?

Answer 1

The isolation involves three steps:

1. Preparation and clearing of a bacterial lysate
2. Absorption of DNA into a silica membrane
3. Washing and elision of plasmid DNA

Question 2

What is does preparation for this lab entail?

Answer 2

• resuspension of the cells
• alkaline lysis of bacteria
• Lysate neutralized and adjusted to high salt binding conditions
• Centrifugation to pellet cell debris thereby producing a clear lysate

Question 3

What is used to resuspend the buffer?

Answer 3

• A resuspension buffer (Tris Cl, EDTA)
• Contains RNAse A
• Contains lyaseBlue

Prevents clumps

Question 4

What is lyseblue?

Answer 4

A color indicator or visual identification system

It’s color contrast prevents common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris

Question 5

What does lysesblue indicate when colorless vs brownish?

Answer 5

If the suspension contains localized regions of colorless solution or brownish cell clumps are still visible, continue mixing the solution until a homogenous blue suspension should is observed

Note: Lyseblue isn’t required to successfully perform plasmid preparation

Question 6

What does the Alkaline Lysis buffer contain?

Answer 6

Alkaline lysis buffer

Contains NaOH/SDS in the presence of RNAse A

-SDS solubilizes the phospholipid and protein components of the cell membrane, leading to lysis and release of the cell contents while the alkaline conditions denature the chromosomal and plasmid DNAs, as well as proteins

Question 7

What is the neutralization buffer?

Answer 7

A colorless solution after the addition of buffer N3 indicates that the SDS from the lysis buffer has been effectively precipitated

-High salt concentration facilitates the precipitation of denatured proteins, chromosomal DNA, cellular debris, and SDS upon centrifugation

Question 8

What is N3?

Answer 8

It is. Important that the solution is thoroughly but gently mixed (NOT VORTEXED) to ensure complete precipitation

Question 9

Describe DNA Separation

Answer 9

Separation of plasmid from chromosomal DNA is based on co-operation of the cell wall-bound chromosomal DNA with insoluble complexes containing salt, detergent, and protein. Plasmid DNA remains in the clear supernatant. Vigorous treatment during the lysis procedure will shear the bacterial chromosome, leaving free chromosomal DNA fragments in the supernatant

Question 10

What is adsorption?

Answer 10

Silica gel membranes used for selective adsorption of DNA in high-salt buffer

RNA, cellular proteins, and metabolites are essentially removed in the flow through

Question 11

Give the procedure for washing/elution(PE/EB)?

Answer 11

Washing steps remove endonucleases that can degrade the plasmid DNA

Washing also removes salts from the high-salt buffer

Plasmid DNA is eluted from the membrane using low salt buffer or water

Elution is pH dependent (max @ 7.0-8.5)

Question 12

How is quantification performed?

Answer 12

Concentration determined by UV spectrophotometry

-nitrogenous bases have max. Absorbance at 260nm

DNA conc. (ug/ml)= OD260 X Dilution factor X( 50 ug/ml 1 OD unit)

Question 13

What should be done the day before this lab?

Answer 13

One day prior to the lab, 2-4 transformed bacterial colonies from one of your plates (lab #1) was inoculated in a culture of LB broth containing 100 ug/ml of ampicillin. The culture was allowed to grow overnight in a shaking water bath at 37 degrees Celsius

Question 14

Explain the preparation of cells

Answer 14

1. Using the 200-1000uL pipettor, fill one of the 1.5 mL micro tubes with bacterial culture
2. Pellet the bacterial cells by Centrifugation at 8000 rpm for 3 minutes using one of the available microCentrifugation
3. Gently pour the supernatant into the waste breaker being careful not to dislodge the pellet of bacterial cells (If necessary use a Kimwipe to remove traces of liquid medium)
4. To the tube containing the bacterial cell pellet add 250 uL of buffer P1. Close the tube
5. Resuspend the bacterial cells in the buffer P1* gently vortex if the cells using the vibrating vortex apparatus (alternatively you can tap or flick the tube). Ensure that the cells are completely resuspended and there are no clumps.
   * ensure that the LyseBlue particles are completely dissolved prior to use

Question 15

What is the procedure to perform alkaline lysis of cells?

Answer 15

6. To the tube containing the resuspended bacteria, add 250 uL of buffer P2
7. Mix thoroughly by inverting the tube 4-6 times (DO NOT VORTEX)
8. If necessary, continue inverting the tube until the solution becomes viscous with a homogenous blue color. (There should be no localized colorless regions or brown clumps)

Do not allow the lysis reaction to proceed more than 5 minutes

9. To the tube containing the cleared bacterial lysate, add 350 uL of buffer N3
10. Mix immediately and thoroughly by inverting the tube 4-6 times. The solution should become clear,
11. Centrifuge the tube for 10 minutes at 13,000 rpm in the microcentrifuge. This will result in the formation of a compact white pellet

Question 16

Explain plasmid purification of DNA

Answer 16

12. Using the 200-1000 uL pipetted with a tip, gently remove the supernatant formed in step #11 and apply it to the spin column. Be sure that the bottom microcentrifuge tube is attached to the spin column before doing this step.
13. Centrifuge the spin column with the bottom microcentrifuge tube attached at full speed for 30-60 seconds
14. Remove the spin column from the microcentrifuge tube and discard the flow through solution.
15. Replace the spin column onto the microcentrifuge tube
16. Wash the spin column by adding 750 uL of buffer PE to the spin column
17. Centrifuge the spin column/microcentrifuge tube at full speed for 30-60 seconds
18. Remove the spin column from the microcentrifuge tube and discard the flow through solution

Question 17

Explain plasmid purification of DNA pt. 2

Answer 17

19. Replace the spin column onto the empty microcentrifuge tube and centrifuge for an additional 30-60 seconds to remove residual wash buffer
20. Place the spin column in a clean 1.5 ml microcentrifuge tube
21. To elute the plasmid DNA, add 50 uL of Huffer EB to the spin column. Be sure to add the buffer directly to the spin column membrane
22. Let stand for 1 minute
23. Centrifuge at full speed for 1 minute
24. Discard the spin column and keep the microcentrifuge tube containing the eluded plasmid DNA

Question 18

What are the steps of determine the concentration of plasmid DNA?

Answer 18

1. Add 40 uL of your plasmid DNA solution obtained from step 24 to the microcentrifuge containing 960 uL of water (labelled ‘DNA’). This will result in a 25 fold dilution of DNA sample.
2. Mix thus solution by using the vibrating vortex or shaking to ensure that the DNA is uniformly mixed into the water
3. Take the diluted DNA to the research lab for further analysis. You will need a pen and paper to write down the absorbance value of your DNA sample.
4. After measuring the absorbance of your DNA samples at 260 Nn wavelength and obtaining the absorbance value, use the formula shown in the introduction to calculate the concentration of your original plasmid DNA sample.
5. You will also given an absorbance value of you sample at 280 Na wavelength. Determine if your DNA sample is of high purity

4.

Question 19

How do we quantify plasmid DNA

Answer 19

1. Add 40 uL of plasmid DNA solution obtained from step 24 to the microcentrifuge containing 960 uL of water(labeled ‘DNA’). This will result in a 25 fold dilution of your DNA sample
2. Mix this solution by using the vibrating vortex or shaking to ensure that the DNA is uniformly mixed into the water.
3. Take the diluted DNA to the research lab for further analysis

Question 20

How to find out if DNA quantification is pure

Answer 20

OD260= 0.053
OD280= 0.031

OD260/OD280=0.035/0.031= 1.7

1. 65-1.80= good, acceptable range
   
   • higher values indicated more purity