Construction

(Difference between revisions)

Revision as of 08:18, 24 October 2006

Andy:

Gel-extrated I13507 (2005) X/P
Made o/n of I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005) for minipreps
Tested UT3 for temperature and arabinose sensitivity, plated UT2, UT3, DH5a for plate test
Made plates
ligated and transformed UT1 with Q4400 (2005) = UT6, Q3400 (2006) = UT7, and Q4400 (2006) = UT8

To-do List:

Massive To-do List

The following is a guideline. Day-to-day details need to be filled in as we progress. Change colors from red to black as each task is completed.

Construction:

Transform and miniprep I13522 (2006) (amp) from registry DNA-2 15H
Transform and miniprep I13504 (2005/2006) (amp) from registry DNA-1 12D (2005/2006)
Miniprep 2 UT1 for ligation (not urgent)
Ligate UT1 with Q03400, Q04400 (2005/2006) (4 ligations) (Name UT6-9)
If possible, ligate R0011 (2005) with I13507 (2005) in parallel (Name UT10)
Ligate UT6-9 with I13504 (4 ligations) (Name UT11-14)
Ligate UT11-14 with UT10 (4 ligations) (Nam UT15-18)

Testing:

Andy:

Gel-extrated R0011 (2005) S/P (band: 2000 - correct)
Cannot gel-extract I13507 (2005) X/P because there was no more extraction column (bands: 2500-3000, 2000-2500, 750-1000 - correct)
Transformed R0011 (2005), I13522 (2006), I13504 (2005/2006)
Quantitated
- Q3400 (2005) E/X (no band)
- Q4400 (2005) E/X (band: 5000 - correct 120 ng/uL)
- Q3400 (2006) E/X (band: 5000 - correct 24 ng/uL)
- Q4400 (2006) E/X (band: 5000 - correct 120 ng/uL)
- UT1 D E/S (band: 2500-3000 - correct 18 ng/uL)
- UT1 E E/S (band: 2500-3000 - correct 12 ng/uL)
- UT1 F E/S (band: 2500-3000 - correct 30 ng/uL)
o/n of UT3 x 5, UT2 x 1, and DH5a x 4 for temperature, arabinose, and plate testing

To-do List:

Ligate UT1 with Q3400 (2006), Q4400 (2005/2006)
Make o/n of I13522 (2006) for miniprep and plate-test, I13504 (2005/2006), I13507 (2005) for miniprep
Gel-extract I13507 (2005) X/P
Temperature, arabinose, and plate testing
Purchase gel-extraction kit, make Amp plates

Guidelines for Plate-test:

On an Amp plate, make 4 sectors (500 uL?) with varying arabinose (0.02%, 0.2%, 2%, 20%) and making sure they don’t touch each other (otherwise diffusion will happen and they’ll be the same concentration), let the arabinose area dry.
Take o/n of UT3-DH5a and take out 200 uL and add 800 uL of fresh LB and incubate for 1 hr.
Spin down the cells at 3000 RPM for 5 min and remove 800 uL of supernatant. Resuspend cells with the remaining 200 uL and gently spread onto plate

Guidelines for Temperature-test:

Put a constant amount of arabinose (either 0.02% or 0.2% - don’t want TOO much repression) in each dilution.
Grow dilutions at 37C until the absorbance is ~0.6 using the GFP ABS program. Then take some tubes out and put 1 mL into an Eppendorf tube.
Use the white filter caps and put them on each Eppendorf tube (to allow air in, but to protect against contamination)
Set mixer to the equivalent to the shaker in the incubator (remember it’s a smaller tube, so there probably needs to be more force on it to get it to shake).
Set temperature to 22C
Check the absorbance after a couple of hours and prepare the samples when the absorbance matches the absorbance of the tubs in the incubator (these should be ready before the 22C ones are ready)
Hopefully we’ll see a significant difference in fluorescence: cold should fluoresce less than hot.