Team:UIOWA/Experiments
Agarose gels and Agarose Gel Electrophoresis
Introduction
Agarose gel electrophoresis is useful for evaluating DNA quality, separating linear DNA fragments by size, and for estimating relative abundance of specific DNA fragments prior to conducting cloning procedures.
Materials
o 500 ml dedicated flask
o Agarose (depends on 1% agarose gel desired)
o 30mL 1X TAE buffer
o paper towel plug
o Gel Red (keep dark - it's light sensitive)
Procedure
- Assembling Gel Bed
1. Place Gel Bed in between the Gel Bed Holder and lock in place by pressing the Holder Walls together and locking them using the handle.
2. Make sure the Gel Bed Holder is stable by using the Level Tool. Adjust the Holder using the knobs until it is stable.
3. Place Well Comb.
- Preparing the Agarose
4. Add 30mL of 1x TAE Buffer to the flask.
5. Measure and add agarose to the flask according to percentage of gel desired.
· 1% agarose = 0.300g agarose
6. Plug the flask with paper towel and microwave agarose and 1x TAE buffer 1 min on High until just beginning to bubble
7. Swirl to aid in dissolving the agarose, and continue microwaving 10 seconds at a time, until all the agarose is in solution.
8. Discard paper plug and add 1 μl of gel red for every 10 mls of agarose (10 μl per 100 ml).....[to be efficient use 1.5 uL in 30mL]
· Gel Red is light sensitive so do this step fast and keep the gel red covered.
9. Swirl solution.
- Pour the Gel
10. Slowly pour into prepared gel beds (small 7 cm square) [ use 40 mls for the 10 cm rectangular gel beds.]
11. Remove any bubbles (or move them away from the wells) created during pouring using a pipet tip.
12. Cover the gel from light and allow to set at room temperature. (Or store Store in bags or boxes at 4oC in the dark and create a humidified environment by adding TAE-soaked paper towels to the bag or box.)
- Gel Electrophoresis
13. Remove Well Comb and place the Gel Bed to the Electrophoresis Machine. The wells in the gel should be closer to the Negative Terminal.
14. Mix 10-20 uL of DNA Sample with Loading dye (5 ul per 20 uL of sample)
· Mix by pipetting in and out or by lightly vortexing.
15. Carefully load the Sample DNA+Loading Dye into the well. Load 2.5 uL of DNA Ladder into a well.
· Keep track of what each well contains.
16. Run @ 75 V - 120 V
Extraction of DNA fragments from an Agarose Gel
Introduction
Enzyme contamination of DNA samples can interfere with subsequent downstream applications. Therefore DNA fragments from enzymatic reactions and agarose gels often must be "cleaned" or purified prior to further use. QIAquick Kits can be used for highly efficient removal of a broad spectrum of enzymes widely used in molecular biology. The Qiagen Gel Extraction kit allows recovery of fragments as small as 70 bp and as large as 10 kb. Buffer QG in the QIAquick Gel Extraction Kit solubilizes the agarose gel slice and provides the appropriate conditions for binding of DNA to the silica membrane in the purification column. DNA adsorbs to the silica membrane in the presence of high concentrations of salt, while contaminants pass through the column. Impurities are efficiently washed away, and pure DNA is eluted with Tris buffer or water. Buffer QG contains an integrated pH Indicator allowing easy determination of the optimal pH for DNA binding. DNA adsorption requires a pH ≤ 7.5, and the pH Indicator in the buffers will appear yellow in this range. If the pH is >7.5, which can occur if during agarose gel electrophoresis, the electrophoresis buffer had been used repeatedly or incorrectly prepared, or if the buffer used in an enzymatic reaction is strongly basic and has a high buffering capacity, the binding mixture turns orange or violet. This means that the pH of the sample exceeds the buffering capacity of Buffer QG and DNA adsorption will be inefficient. In these cases, the pH of the binding mixture can easily be corrected by addition of a small volume of 3 M sodium acetate*, pH 5.0, before proceeding with the protocol.
Materials
· Buffer QG (yellow at pH less than or equal to 7.5)
· Buffer PE (Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
· Isopropanol (100%)
· a heating block or water bath at 50°C are required.
Procedure
- Cut the fragment out of an agarose gel
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel on a UV box. Important: use eye and skin protection while doing this; the UV irradiation is harmful.
2. Weigh the gel slice in a colorless tube whose weight has been pre-determined.
- Dissolve the agarose
3. Add 3 volumes Buffer QG to 1 volume gel
· Note 1: 100 mg gel ~ 100 μl.
· Note 2: The maximum amount of gel per spin column is 400 mg.
· Note 3: For >2% agarose gels, add 6 volumes Buffer QG.
4. Incubate at 50°C for 10 min or until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel.
5. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix.
- Bind the DNA to a Qiaquick 1column
6. Add 1 gel volume isopropanol to the sample and mix.
7. Place a QIAquick spin column in a provided 2 ml collection tube. Apply the sample to the QIAquick column and centrifuge for 1 min.
8. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 μl, load and spin again.
9. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.
· Note 1: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt- ended ligation), let the column stand 2–5 min after addition of Buffer PE.
10. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
· Note 2: This seems trivial and is easy to forget, but it is very important to remove all residual buffer.
- Elute the DNA from the column
11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min.
· Note 1: For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.
· Note 2: After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
- Evaluate Recovery
13. Option 1: If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel. This, of course, uses up your precious sample, but is sometimes necessary to determine whether your DNA has been recovered and whether what was recovered is high quality.
14. Option 2: Alternatively, use the nanospec to get a DNA concentration.
E. coli competent cells
Introduction
This protocol uses cold temperature incubation and commercial (proprietary) competence buffer to maximize the number of transformants/microgram of DNA DH5 alpha cells are a good all-purpose E. coli strain that will be useful for our cloning projects.
Materials
·
o Frozen stock of the desired bacterial strain
o 2x Zymo Research Wash Buffer
o 2x Zymo Research Competent Buffer
o Zymo Research Dilution Buffer
o Spectrophotometer
o Environmental shaker at 18-23oC
Procedure
- Preparation of Z- Competent Cells
1. Start 2 ml E. coli culture (HB101, DH5 alpha) in LB early in the morning
2. At 4-5 PM, inoculate 50 ul saturated culture into 50 ml LB in a 500 ml culture flask. Shake culture ovn (180 rpm) at 18-23 ° until the OD 600nm is 0.4 - 0.6. (18-22 hrs).
- Prepare for Harvesting the Cells
3. Prepare 5 ml of 1X Wash Buffer by adding 2.5 ml Dilution Buffer to 2.5 ml of Wash Buffer (2X Stock).
4. Prepare 5 ml of 1X Competent Buffer by adding 2.5 ml Dilution Buffer to 2.5 ml of Competent Buffer (2X Stock).
5. Store the 1X buffers on ice prior to use.
- Harvesting the Cells
6. Transfer the 50 ml culture from flask to 2 50 ml cent tubes on ice. After 10 minutes, pellet the cells by centrifugation at 4,000 rpm for 10 minutes at 0 - 4°C.
7. Remove the supernatant and resuspend the cells gently in 5 ml ice-cold 1X Wash Buffer.
8. Re-pellet the cells as in Step 4.
9. Completely remove the supernatant and gently resuspend the cells in 5 ml ice-cold 1X Competent Buffer.
10. Aliquot (on ice) 25-100 ul of the cell suspension into sterile microcentrifuge tubes. Cells are now ready for transformation with DNA or can be stored at -70/-80°C for transformation at a later time.
