Team:Minnesota/Experiments
Team:Minnesota
#Minnesota 2018
Experiments:
PCR (Polymerase chain reaction)
Protocol (50 uL system):
1. Add 33uL of dd H2O
2. Add 10uL of Q5 reaction buffer
3. Add 1uL of dNTPs
4. Add 2.5uL of forward primer and 2.5uL of reverse primer
5. Add 1uL of DNA template
Thermocycling Conditions for the PCR:
1: Initial Denaturation: 98-degree Celsius for 30 seconds.
2: 98-degree Celsius for 10 seconds
55 degree celsius for 30 seconds
72 degree celsius for 1:30 seconds
Repeat step two for 34 cycles.
3: Final Extension: 72 degree celsius for 2 minutes.
4: Hold in 6 degree celsius.
DNA gel electrophoresis:
Gel making protocol:
1. Add 200 mL 1xTAE buffer to the Erlenmeyer flask.
2. Add 1% of LE Agarose made by GeneMate (Lot#1193C519).
3. Heating with microwave for 3 minutes covered with tinfoil.
4. Add 80 uL EtBr to the solution. Final concentration is 0.62 ug/L.
5. Pour the solution into the gel template and using 10x comb. Stay for cooling.
Gel Electrophoresis protocol:
1. Add 25 uL of each PCR products to the PCR tube.
2. Add 5 uL 10x Fast Digest Green buffer to each PCR product. Labeled as #1~#9.
3. Add 10 uL Quick Load 2-log ladder into an empty well as the DNA length ladder.
4. Running the gel for 45 min at 120V.
Extracting DNA from gel protocol:
1. Excise the DNA fragment from the agarose gel using a razor blade under Blue light.
2. Place DNA fragment in 1500 uL centrifuge tube.
3. Add 3 volumes of ADB to each tube (100 uL gel volume, 300 uL of ADB Buffer was added)
4. Incubate at 55 degree Celsius until the gel dissolve
5. Transfer the solution to a Zymo PCR DNA spin column with collection tube at the bottom and centrifuge it at 10000 rcf for 60 second
6. Discard the flow through, and add 200 ul of DNA wash buffer to each tube. Centrifuge at 10000 rcf for 30 seconds, repeat this step twice
7. Spin for additional 30 seconds to make sure no wash buffer is left over in the column
8. Add 15 ul of H-pure water to the filter of the spin column, place the spin column on a pre-labeled 1.5 ml microcentrifuge tube, and incubate at room temperature for 10 min 9. Centrifuge the tube for 30 seconds at 10000 rcf.
Transformation:
Protocol (for known DNA using XL1-Blue competent cell):
1. Pre-chill 14-ml BD Falcon polypropylene round-bottom tubes on ice. Preheat SOC medium to 42 °C.
2. Add 10 ul of nuclease free water to iGem plate 5, well 7I containing Plasmid pDU1358 and suspend getting the entirety of the DNA.
3. Centrifuge the tube. (This concentration should be around 200-300 pg/ul)
4. Thaw the super-competent cells on ice. When thawed, add 25 ul of the competent cells into the pre-chilled tube.
5. Add 3 ul of the experimental DNA from the iGem plate into the aliquot of competent cell in the prechilled tube.
6. Incubate tube on ice for 30 minutes.
7. Heat-pulse the tubes in 42 °C water bath for 45 seconds.
8. Incubate tube on ice for 2 minutes.
9. Add 125 ul of SOC medium and incubate tubes at 37 °C for 1 hour w/ shaking at 225-250 rpm.
10. Plate ~ 153 ul (or all of it) of the transformation mixture on LB agar plate containing chloramphenicol. Incubate plates at 37 °C overnight.
Miniprep:
Protocol:
1. Prepare 1500 uL centrifuge tubes according to the number of samples
2. Adjust pipette to 500 uL. Transfer 1500 uL of bacteria solution to each centrifuge tube.
3. Place centrifuge tubes in the centrifuge machine (balance). Spin at 8000 rcf for 1min
4. Carefully discard supernatant using a pipette.
5. Add the remaining bacteria solution to the centrifuge tube.
6. Repeat step 3-5
7. Add 600 uL dd water to each centrifuge tube and resuspend using pipette
8. Add 100 uL lysis buffer (blue) and invert 6-7 times
9. Add 350 uL neutralization buffer (in the fridge) and invert 6-7 times
10. Place centrifuge tubes in the centrifuge machine. Spin at 10000 rcf for 10 min
11. Use pipette to transfer 900 uL of supernatant (Do not damage the pellet) to Zymo spin IIN tube
12. Spin tubes at 10000 rcf for 1 min. Discard the liquid.
13. Add 200 uL endo-wash buffer. Spin at 10000 rcf for 30 sec. Discard the liquid
14. Add 400 uL of Zyppy wash buffer to tubes. Spin at 10000 rcf for 30 sec. Discard the liquid
15. Repeat step 14.
16. Spin at 12000 rcf for 30 sec (dry spin)
17. Discard the bottom tubes and place a 1500 uL centrifuge tube under the Zymo spin IIN tube.
18. Carefully add 50 uL of dd water to the center of the filter of Zymo spin IIN tube.
19. Incubate 5-10 min at room temperature. Spin at 10000 rpm for 30 sec.
Digestion:
Protocol (20 microliter system):
1. Add 2 uL FD.
2. Add 1 uL plasmid.
3. Add 1 uL enzyme (BamHI)/
4. Add dd water until 20 uL.
5. 37°C incubate for 1 hour.
IPTG Experiment set up:
We prepared 8x10 mL of M9 media, and add different volume of IPTG to get 8 different concentrations of IPTG&M9 solution.
The wells of plate were loaded as shown in figure re 1.There wer were total 96 wells in this plate which was separated into 3 groups with 8 different concentrations for LacI 1, LacI 2 and LacI 10.
Figure 1: IPTG plate set up.(IPTG 1000x).
×