Team:Alberta/Notebook May

May 30, 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 37^oC. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too.

May 31, 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 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, 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.

Mike

* working on lab book
* so far have working forms for adding new steps and experiments and navigation of experiments
* today was working on inline editing features and added some styles to the layout
* things to work on: get inline editing to working state, add images to experiment views and possibly image uploading

* added user authentication
* added glossary entry form
* working on encyclopedia entry form

* Working on parts designer javascript applet
o Functioning drag and drop
o Working on adding more features like sequence annotation

May 10, 2010

Construct of Kan Resistance in pSB1C3 Backbone

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

Experiment continues on 11-05-2010.

May 11, 2010

Construct of Kan Resistance in pSB1C3 Backbone

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

May 17, 2010

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

May 18, 2010

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

May 19, 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.

May 20, 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 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.

May 25, 2010

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

Discussed an assembly method. Will start with a polyA tail Anchor oligo which will connect to a polyT tail which is bound to the bead. The anchor will consist of a primer region, a BsaI cut site, a buffer region, and finally, an A prime or B prime end. A byte with an appropriate end can be added to this anchor, and another byte can be added onto that, etc... The last thing to be added to the construct is a Terminator byte consisting of a buffer region, bsaI cut site, primer region and a polyT tail. This second primer sequence must be as uncomplimentary as possible to the anchor's primer site so as to minimize primers binding to each other when PCRing the assembled construct. The primers melting point must be around 65 ^oC. After the construct is assembled on the bead, it is heated off and the polyA tail of the anchor binds to the polyT tail of the terminator and a plasmid is born.

We will purchase NEB oligo dT magnetic and cellulose beads which have a polyT tail (5' to 3') 25 nucleotides long. They are meant for mRNA isolation, but should work fine for our purposes. Used the IDT Analyzer software to determine what length of polyA tail is required for our anchor to have a melting temperature of 30 degrees Celcius (decided to change this melting temp later). This melting temp is sensitive to Na+ and Mg++ concentrations, so referred to ligase buffer and elution buffer for these concentrations.

The BsaI A end should look like this:<---diagram--->

Designed an anchor with a polyA10 tail with a primer (Tm=65.1 ^oC) region and the BsaI cut site with an A' end (ACCC).

May 26, 2010

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

Designed new anchors, ordered oligos. Designed new anchors with larger polyA tail (and without hairpins, or self dimerize-ness).

May 27, 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+)

2.5μL Primer - (PrB psB4A5 ApR-, PrB psB4C5 ChR- or PrB 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

No binding capacity specified in the manual for the beads in terms of mols or mass of DNA or beads. Binding capacity is given in terms of cells (because the beads are meant for mRNA isolation from cells). Will have to quantify binding capacity later.

Derivation of mole matching equation: ssDNA is ~330 grams/(mol*bp).

moles = [concentration X volume] / [(330grams/mol X bp) X length]

moles₁ = moles₂

c₁v₁/[(330g/mol X bp) X l₁] = c₂v₂/[(330g/mol X bp) X l₂]

v₁ = v₂(c₂ X l₁/ c₁ X l₂)

Ordered new primers for anchor:

GCG CGC CCG GTC TCA TGG GTC ACC CTC CC

GGG AGG GTG ACC CAT GAG ACC GGC GCG C[A]₁₂

May 28, 2010

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