A Note About Last Night's Ligation
There seems to have been a power outage last night so the thermocycler was not on and not at 16°C when I came in this morning. The total ligation time was 15 hours and 55 minutes, but I am not sure how much of that time was spent at 16°C and how much of that was spent at room temperature. I have realized that I don't have enough double-digested pQE-80 to re-do the ligation tonight, but I will begin digesting the plasmid and still amplify and transform last night's product.
Amplification of Last Night's Ligation Product
This procedure was done for the wild-type Asc Hb that was ligated with pQE-80-L-Kan last night. The pQE-80 primer was used:
- Mix 29μL of Nuclease-free water, 10μL of 5X Phusion HF Buffer, 1μL of 10mM dNTPs, 2.5μL 10μM forward primer, 2.5μL 10μM reverse primer, and 5.5μL of the ligation product.
- Add 0.5μL of Phusion DNA Polymerase, mix the solution, and centrifuge shortly.
- Put 50μL of wax on top of the mixture and place on the pre-heated block of the thermocycler.
- Start with 30 seconds at 98°C on the thermocycler.
- Cycle through the following 40 times:
- 10 seconds at 98°C
- 30 seconds at 62°C
- 2.5 minutes at 72°C
- A final extension step of 10 min was done at 72°C.
- The thermocycler was then held at 4°C.
Most components for this PCR came from the Phusion® High-Fidelity PCR Kit.
Cutting with HindIII for Cloning
This was done with the pQE-80-L-Kan midiprep from 06/19/12.
- For the pQE-80-L-Kan, mix 27μL of DNA with 5μL of 10x NEBuffer4, 13μL of sterile dH2O, and 5μL of HindIII enzyme.
- Incubate for two hours at 37°C.
- Incubate for 20 minutes at 65°C.
- Store at -20°C.
Running an Analytical DNA Gel
- Make a 1.2% agarose gel (0.3g agarose + 25mL TAE buffer, microwave until boiling, then pour into gel chamber, and place comb for wells).
- When the gel has solidified, add TAE buffer to the chamber until the gel is completely submerged in buffer.
- Load 5μL of DNA ladder into the 1st well.
- Load 5μL of last night's ligation product with 1μL 6x loading buffer into the 3rd well (mix before pipetting into well).
- Load 5μL of this morning's amplified ligation product with 1μL 6x loading buffer into the 5th well (mix before pipetting into well).
- Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel.
- Place the gel in Ethidium Bromide Stain for about 30 minutes.
- View under a UV light.
- Be careful when working with ethidium bromide and UV light.
No bands are seen in the third lane (probably because it's only a very small amount of DNA) but there is a smudge in the fifth lane which indicates something is in it. The transformations being done today will better determine if the ligation worked or not.
- Transformations were done with last night's ligation product, this morning's amplified ligation product, the amplified wildtype Asc Hb ligation from 7/4/12, and the amplified wildtype Asc Hb ligation from 7/10/12.
- Plates were prepared by combining 12.5g of LB with 10g of Agar in 500mL of dH2O. This mixture was autoclaved on a liquid cycle. 500μL of 100mg/mL ampicillin was added to this when it cooled, and plates were then poured.
- Place plastic culture tubes on ice for 15 minutes.
- Place DNA for transformation on ice.
- After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
- Add 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
- Add cells (25uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
- Put tube back in ice for 4 minutes.
- Heat shock at 42°C for 80 seconds.
- Add 100uL of SOC media.
- Shake at 37°C for 1 hour
- Plate 50uL of culture media.
- Incubate overnight at 37°C.