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Team:Alberta/Building Parts
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Revision as of 19:46, 1 June 2010
Building Parts
10-05-2010
PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends.
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
Program:
- 5 min-94oC
- 45 sec-94oC
- 1 min-60oC
- 1 min-72oC
- Repeat 2 through 4 35 times
- 5 min-72oC
11-05-2010
PCR purification of Kanamycin Resistant fragments created 10-05-2010 with Qiagen PCR cleanup kit. Determined concentrations by nanodrop. KanA/B': 101.1ng/μL KanB/A':89.6ng/μL
18-05-2010
Prepared DH5α E.Coli competent cells using the Inoue Method. Transformed DH5α cells with pSB1C3-J04450 and grew overnight at 37oC on Chloramphenicol plates
19-05-2010
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.
20-05-2010
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
27-05-2010
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.
28-05-2010
We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary
30-05-2010
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.
31-05-2010
9/12 of the pSB1C3-KanA/B' Liquid cultures 30-05-2010 were successful and only 1/12 of the pSB1C3-KanB/A' liquid cultures were successful. The pSB4A5, pSB3T5 and pSB4C5 transformations worked. Miniprepped all the liquid cultures that 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, 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 and at Room Temperature for 3 hours.
