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Team:Alberta/Building Parts
From 2010.igem.org
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:5uL 10X PCR buffer | :5uL 10X PCR buffer | ||
:1uL 10uM dNTPs | :1uL 10uM dNTPs | ||
| - | :2uL 50uM MgCl ~2~ | + | :2uL 50uM MgCl~2~ |
:0.5uL Taq polymerase | :0.5uL Taq polymerase | ||
:35.5uL MilliQ H~2~O | :35.5uL MilliQ H~2~O | ||
Revision as of 17:44, 1 June 2010
Contents |
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:
- 1uL p1003 (approx. 1ng)
- 2.5uL prA_p1003+
- 2.5uL prB'_p1003-
- 5uL 10X PCR buffer
- 1uL 10uM dNTPs
- 2uL 50uM MgCl~2~
- 0.5uL Taq polymerase
- 35.5uL MilliQ H~2~O
Program:
- 5 min-94^o^C
- 45 sec-94^o^C
- 1 min-60^o^C
- 1 min-72^o^C
- Repeat 2 through 4 35 times
- 5 min-72^o^C
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/uL KanB/A':89.6ng/uL
18-05-2010
Prepared DH5α E.Coli competent cells using the Inoue Method. Transformed DH5α cells with pSB1C3-J04450 and grew overnight at 37^o^C 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 1uL sample of the Miniprep solutions and digested with NotI at 37^o^C for 1 hour.
Digestion Recipe:
- 1uL Miniprep (between 153.2 ng/ul and 302.7ng/ul determined by nanodrop)
- 1uL NotI
- 1uL 10X ReACT 3
- 7uL 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 37^o^C for 1 hour. Heat inactivated the NotI for 10 minutes at 65^o^C. Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16^o^C for 1 hour then took 15uL to room temperature for 2 hours. Transformed 100uL of DH5α cells with 5uL of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.
Digestion Recipe:
- 1uL Miniprep (302.7ng/ul determined by nanodrop)
- 2uL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/uL)
- 1uL NotI
- 1uL 10X ReACT 3
- 5uL MilliQ
Ligation Recipe:
- 10uL of Digest solution
- 1uL T4 DNA ligase
- 6uL 5X Buffer
- 13uL MilliQ H~2~O
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.
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 37^o^C for one hour and then with EcoRI at 37^o^C 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 DATE, digested with BsaI at 37^o^C for 1.5hours, heat inactivated at 65^o^C 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 16^o^C and at Room Temperature for 3 hours.
