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Team:Alberta/Building Parts
From 2010.igem.org
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Transformed DH5α cells with pSB1C3-J04450 and grew overnight at 37<sup>o</sup>C on Chloramphenicol plates | Transformed DH5α cells with pSB1C3-J04450 and grew overnight at 37<sup>o</sup>C on Chloramphenicol plates | ||
| - | =19-05-2010= | + | ==19-05-2010== |
From the transformation of DH5α cells with pSB1C3-J04450 performed on [[#18-05-2010|18-05-2010]], we took 4 distinct colonies, streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures. | From the transformation of DH5α cells with pSB1C3-J04450 performed on [[#18-05-2010|18-05-2010]], we took 4 distinct colonies, streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures. | ||
| - | =20-05-2010= | + | ==20-05-2010== |
Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5α cells with pSB1C3-J04450 from [[#19-05-2010|19-05-2010]]. Took a 1μL sample of the Miniprep solutions and digested with NotI at 37<sup>o</sup>C for 1 hour. | Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5α cells with pSB1C3-J04450 from [[#19-05-2010|19-05-2010]]. Took a 1μL sample of the Miniprep solutions and digested with NotI at 37<sup>o</sup>C for 1 hour. | ||
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| - | =27-05-2010= | + | ==27-05-2010== |
Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from [[#11-05-2010|11-05-2010]] and pSB1C3 from [[#20-05-2010|20-05-2010]] with NotI at 37<sup>o</sup>C for 1 hour. Heat inactivated the NotI for 10 minutes at 65<sup>o</sup>C. Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16<sup>o</sup>C 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. | Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from [[#11-05-2010|11-05-2010]] and pSB1C3 from [[#20-05-2010|20-05-2010]] with NotI at 37<sup>o</sup>C for 1 hour. Heat inactivated the NotI for 10 minutes at 65<sup>o</sup>C. Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16<sup>o</sup>C 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. | ||
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Also transformed pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450 from the 2010 biobrick parts into DH5α cells. | Also transformed pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450 from the 2010 biobrick parts into DH5α cells. | ||
| - | =28-05-2010= | + | ==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 | 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= | + | ==30-05-2010== |
From the transformation of DH5α cells with pSB1C3-KanR performed on [[#28-05-2010|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 37<sup>o</sup>C. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too. | From the transformation of DH5α cells with pSB1C3-KanR performed on [[#28-05-2010|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 37<sup>o</sup>C. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too. | ||
| - | =31-05-2010= | + | ==31-05-2010== |
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 transformations worked. Miniprepped all the liquid cultures that worked. | 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 transformations worked. Miniprepped all the liquid cultures that worked. | ||
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Performed a restriction digest of an aliquot of the pSB1C3-KanA/B' and pSB1C3-KanB/A' minipreps. Digested with XbaI at 37<sup>o</sup>C for one hour and then with EcoRI at 37<sup>o</sup>C for one hour. Ran a 1% agarose gel of the digests to determine the orientation of the KanR fragments. | Performed a restriction digest of an aliquot of the pSB1C3-KanA/B' and pSB1C3-KanB/A' minipreps. Digested with XbaI at 37<sup>o</sup>C for one hour and then with EcoRI at 37<sup>o</sup>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 [[#11-05-2010|11-05-2010]], digested with BsaI at 37<sup>o</sup>C for 1.5hours, heat inactivated at 65<sup>o</sup>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<sup>o</sup>C and at | + | KanA/B' and KanB/A' fragements PCRed on [[#11-05-2010|11-05-2010]], digested with BsaI at 37<sup>o</sup>C for 1.5hours, heat inactivated at 65<sup>o</sup>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<sup>o</sup>C. |
| + | |||
| + | ==01-06-2010== | ||
| + | |||
| + | KanA/B' and KanB/A' fragements PCRed on [[#11-05-2010|11-05-2010]], digested with BsaI at 37<sup>o</sup>C for 1.5hours, heat inactivated at 65<sup>o</sup>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 for 3 hours at 21<sup>o</sup>C. | ||
| + | |||
| + | Set up liquid cultures from plates streaked on [[#30-05-2010|30-05-2010]] | ||
| + | |||
| + | ==02-06-2010== | ||
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{| style="color:#1b2c8a;background-color:#FFFF33;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" | {| style="color:#1b2c8a;background-color:#FFFF33;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" | ||
Revision as of 18:02, 3 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.
01-06-2010
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 for 3 hours at 21oC.
Set up liquid cultures from plates streaked on 30-05-2010
02-06-2010
| Building Parts | Testing Parts | Assembly Method | Competent Cells | Plates | Kit Manual | Software | Notebook |
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