User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/21

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Transformation Results

The transformations that grew overnight last night look to all have colonies. The colonies are very small and grouped together so I will allow them to continue to grow at 37°C for the day. Tonight I'm going to try plating some of the new transformations diluted so the colonies might be more separated.

Running an Analytical DNA Gel

  1. 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).
  2. When the gel has solidified, add TAE buffer to the chamber until the gel is completely submerged in buffer.
  3. Load 5μL of DNA ladder into the 1st well.
  4. Load 5μL of the M8S/M33S/M103S/A71M/L40M PCR product from yesterday with 1μL 6x loading buffer into the 2nd well (mix before pipetting into well).
  5. Load 5μL of the wild-type Asc Hb and pQE-80-L-Kan ligation (5μL insert, 1μL vector) from yesterday with 1μL 6x loading buffer into the 4th well (mix before pipetting into well).
  6. Load 5μL of the wild-type Asc Hb and pQE-80-L-Kan (10μL insert, 2μL vector) ligation from yesterday with 1μL 6x loading buffer into the 5th well (mix before pipetting into well).
  7. Load 5μL of the triple mutant (M8S/M33S/M103S) Asc Hb and pQE-80-L-Kan ligation (5μL insert, 1μL vector) from yesterday with 0.5μL 6x loading buffer into the 7th well (mix before pipetting into well).
  8. Load 5μL of the triple mutant (M8S/M33S/M103S) Asc Hb and pQE-80-L-Kan ligation (10μL insert, 2μL vector) from yesterday with 0.5μL 6x loading buffer into the 8th well (mix before pipetting into well).
  9. Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel
  10. Place the gel in Ethidium Bromide Stain for about 30 minutes
  11. Move the gel to TAE buffer to destain for about 20 minutes
  12. View under a UV light
    • Be careful when working with ethidium bromide and UV light.


Image:Photo_(17).JPG

There are no bands (besides the ladder bands) on this gel. This means the PCR from yesterday and the ligations from last night probably didn't work well, if at all. I don't really want to do a transformation with no DNA, so I'll re-run both of these procedures today and do the transformation tomorrow.

Making Media for Expression

  • Make 4 1L LB broths: each is 25g LB (w/o NaCl) + 10g NaCl with 1L of dH2O
  • Make 4 25mL LB broths: each is 0.63g LB (w/o NaCl) + 0.25g NaCl with 25mL of dH2O
  • LB/agar Plates: 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar + 250mL dH2O, autoclave liquid cycle, cool to about 60°C, add 250μL 100mg/mL amicillin, pour plates, store at 4°C
    • Autoclave all of these broths
    • Also, autoclaved a dry cycle today.

Split PCR for L40M Mutation

Using a modified version of 2-stage QuikChange the L40M mutation should be inserted into the Colony 3 miniprep product from yesterday (M8S/M33S/M103S/A71M). Changing colonies for this PCR in case the other one had something wrong with it and if why there was no bands on the DNA gel from this morning. Hopefully this miniprep has the proper mutation (will be sequenced next week).

Protocol

First Stage

1st stage
Tube sterile H2O Pfu Buffer Col. 3 M8S/M33S/M103S/A71M mutated hemoglobin For primer (L40M f) Rev primer (L40M r)dNTPs Pfu Turbo wax
(10X) (12.5 ng/μL) (12.5 ng/μL) (10 mM ea) (2.5 U/μL)
Experimental-forward 30 μL 5 μL 2 μL 10 μL 0 μL 2 μL 1 μL 50 μL
Experimental-reverse 30 μL 5 μL 2 μL 0 μL 10 μL 2 μL 1 μL 50 μL
  • Combine all components of each mixture in sterile PCR tubes, except for Pfu Turbo and wax, mix well, but gently
  • Add Pfu Turbo and mix gently with pipet
  • Add wax on top of mixture and place on thermoclycler
  • 30" @ 95°C
  • Cycle through the following six times:
      1. 30" @ 95°C
      2. 1' @ 55°C
      3. 5' @ 68°C
  • Remove from thermocycler, and pipet wax off of each reaction mixture
  • Begin second stage

Second Stage

2nd stage
Tube 1st stage-forward 1st stage-reverse Pfu Turbo wax
(2.5 U/μL)
Experimental 25 μL 25 μL 1 μL 50 μL
  • Combine mixtures from first stage and mix together gently with pipet, then add Pfu Turbo and mix again gently with pipet
  • Add wax on top of mixture and place on thermoclycler
  • 30" @ 95°C
  • Cycle through the following 20 times:
      1. 30" @ 95°C
      2. 1' @ 55°C
      3. 5' @ 68°C
  • 15' @ 68°C
  • hold @ 4°C

Start Cultures for Miniprep

Grow the transformations from last night (3 colonies of each), each in 10mL of LB with 10μL of 100mg/mL ampicillin at 37°C at 250rpm overnight.

Start Wildtype Hemoglobin Expression

  1. Add 25μL of 100mg/mL ampicillin to each 25mL LB culture (4 of them).
  2. Take a sterile wooden stick and scrape the glycverol stock and swirl in each LB/Amp culture.
  3. Grow overnight at 37°C at 250rpm.



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