Construction
From 2006.igem.org
Line 7: | Line 7: | ||
'''Charles, Nick:''' | '''Charles, Nick:''' | ||
- M9 Test – 0% - 2% Arabinose in UT3 and DH5a (control), temperature test at 0.2% Arabinose, IPTG verification at 0% and 2% arabinose: | - M9 Test – 0% - 2% Arabinose in UT3 and DH5a (control), temperature test at 0.2% Arabinose, IPTG verification at 0% and 2% arabinose: | ||
- | + | ||
+ | |||
+ | |||
+ | |||
+ | - Dilute o/n of UT3 and DH5a in 1:50 o/n to LB ratio in appropriate arabinose/IPTG concentrations | ||
– incubate 3 hrs | – incubate 3 hrs | ||
- | + | - Resuspend in 50:50 M9:LB (including arabinose/IPTG) and divided 0.2% arabinose for temperature | |
test – incubate for 3 hrs | test – incubate for 3 hrs | ||
- | + | - Resuspend in M9 media – incubate for 5 hrs | |
- First 3 hrs without signalling conditions, then latter 2 hrs with conditions | - First 3 hrs without signalling conditions, then latter 2 hrs with conditions | ||
- | + | - Read with fluorometer (PBS wash not required) | |
- Ligated I0500 B (2005) + I13507 (2005) / I13504 (2005/2006) | - Ligated I0500 B (2005) + I13507 (2005) / I13504 (2005/2006) | ||
- Made o/n of UT9, UT10, UT11 ABC | - Made o/n of UT9, UT10, UT11 ABC |
Revision as of 08:06, 2 November 2006
< Aug11-Sep7 | Sep8-14 | Sep15-26 | Sep27-Oct3 | Oct4-Oct13 | Construction >
Contents |
October 27, 2006
Charles, Nick: - M9 Test – 0% - 2% Arabinose in UT3 and DH5a (control), temperature test at 0.2% Arabinose, IPTG verification at 0% and 2% arabinose:
- Dilute o/n of UT3 and DH5a in 1:50 o/n to LB ratio in appropriate arabinose/IPTG concentrations – incubate 3 hrs - Resuspend in 50:50 M9:LB (including arabinose/IPTG) and divided 0.2% arabinose for temperature test – incubate for 3 hrs - Resuspend in M9 media – incubate for 5 hrs - First 3 hrs without signalling conditions, then latter 2 hrs with conditions - Read with fluorometer (PBS wash not required)
- Ligated I0500 B (2005) + I13507 (2005) / I13504 (2005/2006) - Made o/n of UT9, UT10, UT11 ABC - Made o/n of DH5a and DH5a-z1
To-Do List: - Check lengths of I0500 (2006), Q04400 (2005/2006), Q03400 (2006) - Miniprep and check lengths of UT9, UT10, UT11 – if correct, wait for functionality test before transforming into cells - Make competent cells (DH5a and DH5a-z1) - Make Amp and Kan plates
October 26, 2006
Andy, Natalie, Charles, Tara, HoKwon:
- Ligated and transformed UT6, UT7 with I13504 (2005/2006) (Name UT10, UT11) (Quantitation showed correct band for all)
- Ligated and transformed R0011 (2005) with I13507 (2005) (Name UT9) (Quantitation showed correct band for all)
- Made o/n of UT3 x 3, DH5a for testing
- Quantitated I0500 (2005) AB to be ligated to I13504 (2005/2006) (Band: 6000 - roughly correct. A 20 ng/uL B 300 ng/uL)
To-do list:
- Make o/n of UT9, UT10, UT11 for miniprep
- Ligate I0500 (2005) with I13504 (2005/2006)
- M9 testing of UT3
October 25, 2006
Andy:
- Minipreppred UT6, UT7, UT8
- Gel-extracted (only for correct bands)
- UT6 S/P (band: 8000-10000 correct) -- 80 ng/uL
- UT7 S/P (band: 8000-10000 correct) -- 50 ng/uL
- UT8 S/P (band: 6000 incorrect)
- I13504 (2005) X/P (band: 750-1000, 1500-2000 correct) -- 140 ng/uL
- I13504 (2006) X/P (band: 750-1000, 1500-2000 correct) -- 90 ng/uL
- Made o/n of UT2 x 3, UT3 x 3, DH5a for testing
To-do list:
- Quantitate and ligate UT6, UT7 with I13504 (2005/2006)
- Quantitate and ligate R0011 (2005) with I13507 (2005)
- M9 testing of UT2, UT3
October 24, 2006
Melinda:
- Minipreppred and length-checked (unsuccessful) I13522 (2006), I13504 (2005/2006) AB, R0011 (2005)
- Made o/n of UT6-8 for miniprep
October 23, 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:
- Quantitate and Ligate R0011 (2005) with I13507 (2005)
- Miniprep and length-check I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005)
- Make multiple o/n of UT6-8 for miniprep
October 22, 2006
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:
- Temperature-test and Arabinose/IPTG-test UT3 (See below for protocol)
- Plate-test UT2, UT3, DH5a, UT13, I13522 (See below for protocol)
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.
[http://2006.igem.org/University_of_Toronto_2006 Home]
October 19, 2006
Melinda:
- Miniprepped Q04400 (2005/2006) and Q04510 (2005/2006)
- Made o/n of Q03400 (2005/2006), (3) UT2-DH5a, (2) DH5a, and (1) DH5a/DH5a-z1 for freezer stock and fresh plate
To-Do List:
- Miniprep Q03400 (2005/2006)
- Repeat Oct 18 test, except with GFP and proper controls
- Digest and check J04450 (W), Q03400, Q04400, and Q04510 (2005/2006). If correct, move on with gel extraction and ligation.
- Make 20% Arabinose, and 2 blank agar plates
[http://2006.igem.org/University_of_Toronto_2006 Home]
October 18, 2006
Andy, Konstantin:
- Transformed and plated Q03400 (2005) (2006)
- Gel-extracted UT1 DEF with EcoRI/SpeI (2500-3000, 5000, 8000), 8000 band bright, digestion appeared incomplete
- Made A, K, AK plates
- Made o/n of Q04400 (2005) (2006) Q04510 (2005) (2006)
- Tested UT3 in DH5a with 0%-2% arabinose
To-Do List:
- Make o/n of Q03400 (2005) (2006)
- Miniprep Q04400 (2005) (2006) and Q04510 (2005) (2006)
- Replate UT1 with miniprep from a good conlony
- Miniprep Q04400 (2005) (2006) Q04510 (2005) (2006) and check to see if bands are correct. If so, digest with EcoRI/XbaI and gel extract
- Ligate with UT1
[http://2006.igem.org/University_of_Toronto_2006 Home]
October 17, 2006
Melinda:
- Double Digetsed J04450 (W), UT1 DEFG with XbaI/SpeI using Buffer 2
- Results:
- J04450 (W) (1 band)
- UT1 D (5000 – 4000, 3000 – 2500)
- UT1 E (2 bands)
- UT1 F (2 bands)
- UT1 G (2 bands)
- Transformed DH5a cells with Q04400 (2005/2006), Q04510 (2005/2006)
- Made o/n of DH5a and (3 vials) of DH5a UT3
Note – Change in protocol:
- After incubation for 1 hour, centrifuge tubes for 5 min @ 3000 RPM to pellet cells
- Remove 800 uL of supernatant
- Resuspend pellet with remaining 200 uL
- Spread on plate and wait ~15-20 min.
[http://2006.igem.org/University_of_Toronto_2006 Home]
October 16, 2006
Melinda:
- Checked parts, including the relevant parts from Waterloo (W):
- I0500 (W): (5000 – 4000, 1500 – 1000) – correct!
- J04450 (W): (2500 – 2000) – plasmid is correct, but no part?
- J06801 (W): (8000 – 6000) – not correct!
- E0240 (W): (4000 – 3500, 3500 – 3000, 1000 – 750) – part is correct?
- I12006 (W): (5000 – 4000, 2500 – 2000) - plasmid is correct, undigested plasmid?
- UT2 2: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
- UT2 4: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
- UT3 7: (3500 – 3000, 2500 – 2000) – correct!
- Viewed cells induced by IPTG under microscope and verified the functionality of UT2/UT3 in DH5a and DH5a-z1
To-Do List:
- Repeat Double Digest of J04450 (W) and UT1 ABCD with XbaI/SpeI (Run undigested plasmids beside each double digest)
- Make more Amp plates
- If digest is successful, transform DH5a cells with the successful parts
- Find tetR replacements for cI and ligate those parts together.
[http://2006.igem.org/University_of_Toronto_2006 Home]
October 15, 2006
Charles:
- Made o/n of the newly plated UT2 2/4 and UT3 7 as well as UT2/UT3 freezer stock
To-Do List:
- Make sure the cells fluoresce by diluting in IPTG
[http://2006.igem.org/University_of_Toronto_2006 Home]
October 14, 2006
Charles, Jovan:
- Mini-prepped UT2 2/4 and UT3 7
- Transform UT2 2/4 and UT3 7 into DH5a-z1 and DH5a cells
- Continue with the IPTG test for the rest of the colonies that we weren’t able to test yesterday
To-Do List:
- Measure absorbance spectrum of Arabinose
- Make o/n of working UT2/UT3 for repeat of IPTG test.
- Look into using tetR and tet pL instead of cI and Prm+
[http://2006.igem.org/University_of_Toronto_2006 Home]