User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/22: Difference between revisions

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==Miniprep Growths==
==Miniprep Growths==
None of the cultures started for the minipreps had any growth last night (the "colonies" were very small). This means no minipreps will be done today. Two colonies did however appear on the NovaBlue + M103S transformation plate from [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/20|two days ago]]. The plate was just left at room temperature overnight.
None of the cultures started for the minipreps had any growth last night (the "colonies" were very small). This means no minipreps will be done today. Two colonies did however appear on the NovaBlue + M103S transformation plate from [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/20|two days ago]]. The plate was just left at room temperature overnight.


==DNA ligation with T4 DNA Ligase==
==DNA ligation with T4 DNA Ligase==

Revision as of 07:40, 22 June 2012

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Miniprep Growths

None of the cultures started for the minipreps had any growth last night (the "colonies" were very small). This means no minipreps will be done today. Two colonies did however appear on the NovaBlue + M103S transformation plate from two days ago. The plate was just left at room temperature overnight.

DNA ligation with T4 DNA Ligase

This will be done with both the wildtype Asc Hb double-digested insert and the M8S/M33S/M103S Asc Hb double-digested insert. The procedure is based off of the protocol on the NEB website.

  1. Mix 2μL of 10x T4 DNA Ligase Buffer, 5μL of the double-digested pQE-80-L-Kan vector, 10μL of the wildtype insert, 2μL of nuclease free H2O, and 1μL of T4 DNA Ligase.
  2. Mix 2μL of 10x T4 DNA Ligase Buffer, 5μL of the double-digested pQE-80-L-Kan vector, 6μL of the triple mutant insert, 6μL of nuclease free H2O, and 1μL of T4 DNA Ligase.
  3. Place at room temperature for 10 minutes.
  4. Place on ice.

My calculations yesterday led me to a 5:1 volume ratio, but I thought about it again and here is my reasoning for for today for a 3:1 insert to vector molar ratio:

  • Since the vector is about 4700bp and the insert is about 450bp, then the molecular weight of the vector is probably about 10 times bigger than the molecular weight of the insert.
  • I then reasoned that the molarity ratio for the wildtype insert to the vector was about 1.5:1 because of the concentration calculated and the relative molecular weights.
    • This seems to mean that the ratio of volumes for insert to vector should be 2:1 for the wildtype insert
  • I also reasoned that the molarity ratio for the triple mutant insert to the vector was about 2.6:1.
  • I could basically stay close to a 1:1 ratio for the volume ratio, but the real volume ratio would be about 1.15:1
  • I was also aiming for about 50ng of each, but since the molar ratio seems to be most important, I will only aim for 50ng of the vector. This means there will be more vector than insert in terms of mass, but not molarity.

Running an Analytical DNA Gel

Transformations

  1. Place plastic culture tubes on ice for about 15 min.
  2. Place DNA for transformation on ice.
  3. After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
  4. Add 1, 2, or 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  5. Add cells (30uL, 40uL, or 50uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
  6. Put tube back in ice for 4 minutes.
  7. Heat shock at 42°C for 80 seconds.
  8. Add 100uL of SOC media.
  9. Shake at 37°C for 1 hour.
  10. Plate 50uL of culture media on LB/amp plates (prepared yesterday: 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 plate, store at 4°C).
  11. Incubate inverted overnight at 37°C.



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