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Team:Alberta/Notebook May
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Revision as of 16:54, 9 July 2010
May 2010
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The lab notebook chronicles our journey in the creation of the Genomikon kit. Many paths were woven together in space and time to reach this finished masterpiece. To help you navigate through these trials with us we have laid out our notebook in a layered fashion. This page gives a sketch of each project and how it interacts with each other. Then follow the links to a projects page for time line of the major landmarks and accomplishments. If you require more details on the project the links within that page will take you to our day-by-day work log.
The Building Parts project was responsible to first build a plasmid (plasmid 01)that contained our own specialized prefix and suffix nested inside of the standard BioBrick prefix and suffix. After plasmid 01 existed we inserted the CcdB gene (the "death" gene) between our prefix and suffix removing the gene for Kanamycin resistance (plasmid 02). Plasmid 02 is fantastic base plasmid from which we are able to amplify any part at all because it provides a positive selection marker when transformed into DH5α. At this point we were able to make parts en masse to put in our kit. After obtaining a particular part in a plasmid we PCRed the part and digested it ready to use in Assembly or to Test the plasmid.
Before we were able to test parts we created 2 base testing plasmids (vector 01 and vector 02). Vector 01 is designed to test Open Reading Frame parts, or parts that code for proteins. The part is flanked by a promoter and the start codon on one side and a stop codon and terminator on the other. Vector 02 is designed to test linker parts, or parts that control the expression of the Open Reading Frame parts they are next two. In Vector 02 the part is flanked by two distinct reporter genes, that by comparing the relative expression of the 2 reporter genes we can determine the behavior of the linking part.
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Building Parts
From the transformation of DH5α cells with pSB1C3-KanR performed on 28-05-2010, we took 12 distinct colonies of each KanA/B' and KanB/A', streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures with the appropriate antibiotics overnight at 37oC. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too.
Building Parts
9/12 of the pSB1C3-KanA/B' Liquid cultures [[#30-05-2010|30-05-2010]] were successful and only 1/12 of the pSB1C3-KanB/A' liquid cultures were successful. The pSB4A5, pSB3T5 and pSB4C5 liquid cultures worked. Miniprepped all the liquid cultures that worked. However, the streaks on the plates worked.
Performed a restriction digest of an aliquot of the pSB1C3-KanA/B' and pSB1C3-KanB/A' minipreps. Digested with XbaI at 37oC for one hour and then with EcoRI at 37oC for one hour. Ran a 1% agarose gel of the digests to determine the orientation of the KanR fragments.
KanA/B' and KanB/A' fragements PCRed on [[#11-05-2010|11-05-2010]], digested with BsaI at 37oC for 1.5hours, heat inactivated at 65oC for 30 minutes. Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other. Also tried to ligate KanA/B' fragments with KanB/A'. Ligated with T4 DNA ligase overnight at 16oC.
Building Parts
PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends. (Fragments formed called KanR A/B'-Bsa and KanR B/A'-Bsa respectively)
Recipe:
- 1μL p1003 (approx. 1ng)
- 2.5μL prA_p1003+
- 2.5μL prB'_p1003-
- 5μL 10X PCR buffer
- 1μL 10uM dNTPs
- 2μL 50uM MgCl2
- 0.5μL Taq polymerase
- 35.5μL MilliQ H2O
Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-.
Program:
- 5 min-94oC
- 45 sec-94oC
- 1 min-60oC
- 1 min-72oC
- Repeat 2 through 4 35 times
- 5 min-72oC
Building Parts
PCR purification of KanR A/B'-Bsa and KanR B/A'-Bsa created 10-05-2010 with Qiagen PCR cleanup kit.
Determined concentrations by nanodrop. KanA/B'-Bsa: 101.1ng/μL KanB/A'-Bsa:89.6ng/μL
Building Parts
Innoculated 250mL overnight cultures with 10mL,4mL and 2mL of a starter culture of DH5α. Left shaking at 18oC overnight.
Building Parts
Prepared DH5α E.Coli competent cells using the Inoue Method.
Transformed DH5α cells with pSB1C3-J04450 and grew overnight at 37oC on Chloramphenicol plates
Building Parts
From the transformation of DH5α cells with pSB1C3-J04450 performed on 18-05-2010, we took 4 distinct colonies, streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures.
Building Parts
Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5α cells with pSB1C3-J04450 from 19-05-2010. Took a 1μL sample of the Miniprep solutions and digested with NotI at 37oC for 1 hour.
Digestion Recipe:
- 1μL Miniprep (between 153.2 ng/μl and 302.7ng/μl determined by nanodrop)
- 1μL NotI
- 1μL 10X ReACT 3
- 7μL MilliQ
Ran Digestion on a 1% agarose gel to check that the plasmid obtained with what we expected.
Building Parts
Made 1.5mL LB liquid cultures of pSB1C3 from the plate streaked on 19-05-2010 and added chloramphenicol.
Building Parts
Made 3 glycerol stocks of pSB1C3 from overnight made 25-05-2010.
Building Parts
Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from 11-05-2010 and pSB1C3 from 20-05-2010 with NotI at 37oC for 1 hour. Heat inactivated the NotI for 10 minutes at 65oC. Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16oC for 1 hour then took 15μL to room temperature for 2 hours. Transformed 100μL of DH5α cells with 5μL of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.
Digestion Recipe:
- 1μL Miniprep (302.7ng/μl determined by nanodrop)
- 2μL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/μL)
- 1μL NotI
- 1μL 10X ReACT 3
- :5μL MilliQ
Ligation Recipe:
- 10μL of Digest solution
- 1μL T4 DNA ligase
- 6μL 5X Buffer
- 13μL MilliQ H2O
Also transformed pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450 from the 2010 biobrick parts into DH5α cells.
Performed PCR reactions to create parts with antibiotic resistance with negative controls.
PCR Recipe:
- 3μL 10X PCR Buffer
- 1μL 10 uM dNTPs
- 2μL 50 uM MgCl2
- 17.5μL MilliQ H2O
- 0.5μL Taq Polymerase
- 1μL Template (psB4A5-J04450, psB4C5-J04450 or psB3T5-J04450)
- 2.5μL Primer + (PrA psB4A5 ApR+, PrA psB4C5 ChR+ or PrA psB3T5 TR+)
PCR Program:
- 5 min-94oC
- 45 sec-94oC
- 1 min-60oC
- 1 min-72oC
- Repeat 2 through 4 35 times
- 5 min-72oC
Building Parts
We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary
