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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 37^oC. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too.

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 37^oC for one hour and then with EcoRI at 37^oC 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^oC for 1.5hours, heat inactivated at 65^oC 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^oC.

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-94^oC

45 sec-94^oC

1 min-60^oC

1 min-72^oC

Repeat 2 through 4 35 times

5 min-72^oC

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

Innoculated 250mL overnight cultures with 10mL,4mL and 2mL of a starter culture of DH5α. Left shaking at 18^oC overnight.

Prepared DH5α E.Coli competent cells using the Inoue Method.

Transformed DH5α cells with pSB1C3-J04450 and grew overnight at 37^oC on Chloramphenicol plates

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.

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 37^oC 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.

Made 1.5mL LB liquid cultures of pSB1C3 from the plate streaked on 19-05-2010 and added chloramphenicol.

Made 3 glycerol stocks of pSB1C3 from overnight made 25-05-2010.

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^oC for 1 hour. Heat inactivated the NotI for 10 minutes at 65^oC. Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16^oC 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-94^oC

45 sec-94^oC

1 min-60^oC

1 min-72^oC

Repeat 2 through 4 35 times

5 min-72^oC

We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary