Protocols: From Transformations to Ligations
1. If using DNA from Registry, dilute with 9 uL of H2O. If using own DNA, use about 200 ng.
2. Thaw 50uL competent cells on ice.
3. Mix competent cells with 50uL CaCl2 (if competent cells need it).
4. Add 4 uL of Registry DNA, or 200 ng of your own DNA to cells.
5. Place in ice bath for 30 minutes.
6. Heat shock at 42 C for 45-60 seconds.
7. Place in ice bath for 2 minutes.
8. Add 900 uL ice cold, antibiotic free LB to cells.
9. Incubate at 37 C for 90 minutes in a shaking incubator.
10. Dilute as desired, make necessary controls, and plate cells (200 uL per plate).
11. Incubate upside down at 37 C for 18-24 hours, then refrigerate.
1. Pick a single colony off of plate of transformed cells.
2. Mix colony with 5 mL of LB inside culture tube. Use appropriate antibiotic.
3. Place culture tube in mixing incubator at 37 C overnight.
4. Remove from incubator, spin culture tubes at 3000 rpm for 10 minutes. Remove supernatant, then use Qiagen miniprep kit to extract plasmids.
1. Combine the following ingredients:
a. 1 uL of each restriction enzyme to be used.
b. 500-5000 ng of DNA.
c. 5 uL of compatible buffer (if buffer is not compatible with a restriction enzyme,
more of that enzyme should be used).
d. 0.5 uL of BSA.
e. 1 uL of Antarctic Phosphatase.
f. 5 uL of Antarctic Phosphatase Buffer.
g. Enough H2O for 50 uL overall volume.
2. Incubate at 37 C for 2-4 hours (or overnight).
3. Incubate at 80 C for 20 minutes to heat inactivate enzymes.
1. Add 2 volumes ice cold absolute ethanol to sample.
2. Incubate 1 hr at -80°C.
3. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
4. Remove supernatant.
5. Wash with 750-1000 μL room-temperature 95% ethanol.
6. Centrifuge for 10 minutes at 4°C at maximum speed (generally >10000 g at least).
7. Let air dry on benchtop.
8. Resuspend in an appropriate volume of H2O.
There are two options for ligations, 3A and standard. These are more general protocols, as we did not find a specific way to do them with a high rate of success.
1. For these ligations the following are necessary:
a. The prefix part cut with EcoRI and SpeI.
b. The suffix part cut with XbaI and PstI.
c. A construction plasmid with P1010 cut out of it with EcoRI and PstI. The construction plasmid has
to have a different antibiotic resistance than both of the inserted parts.
2. Mix the three together with T4 DNA ligase and ligation buffer.
3. Heat inactivate after certain period of time.
3. Use products to transform competent cells.
4. Plate out the cells on a plate with an antibiotic corresponding to resistance of the construction plasmid.
1. After Ethanol Precipitation of Digested parts, run DNA through gel to extract parts of correct length.
2. Ethanol Precipitate these parts.
3. Add the insert and the backbone together in solution with T4 DNA ligase and ligase buffer.
4. Heat inactivate after certain period of time.
5. Ethanol Precipitate product.
6. Gel extract to select for plasmids of appropriate length.
7. Ethanol precipitate these plasmids.
8. Use them to Transform competent cells.
9. Plate cells with appropriate antibiotics.
Here are copies of our presentations delivered to advisors, faculty, and friends of iGEM:
August Update (August 22nd, 2007):
Synthetic Biology @ Brown (August 10th):
High School Presentation (July 30th):
July Update (July 18th, 2007):
June Update (June 15th, 2007):