Team:Alberta/Building Parts

From 2010.igem.org

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== Building Parts ==
== Building Parts ==
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{| style="color:#1b2c8a;background-color:#0;" cellpadding="0" cellspacing="1" border="0" bordercolor="#fff" width="100%" align="left"
 
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!align="center"|{{ #calendar: title=Team:Alberta |year=2010 | month=05 }}
 
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!align="center"|{{ #calendar: title=Team:Alberta |year=2010 | month=06 }}
 
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!align="center"|{{ #calendar: title=Team:Alberta |year=2010 | month=07 }}
 
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!align="center"|{{ #calendar: title=Team:Alberta |year=2010 | month=08 }}
 
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!align="center"|{{ #calendar: title=Team:Alberta |year=2010 | month=09 }}
 
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=10-05-2010=
=10-05-2010=
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Program:
Program:
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# 5 min-94^o^C
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# 45 sec-94^o^C
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# 1 min-60^o^C
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# 1 min-72^o^C
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# Repeat 2 through 4 35 times
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# 5 min-72^o^C
<!--Image of gel performed that day. 5uL of each PCR reaction, 1uL of 10X loading dye and 4uL MilliQ water in a 1% agarose gel -->
<!--Image of gel performed that day. 5uL of each PCR reaction, 1uL of 10X loading dye and 4uL MilliQ water in a 1% agarose gel -->
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=11-05-2010=
=11-05-2010=
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PCR purification of Kanamycin Resistant fragments created [[#10-05-2010|10-05-2010]] with Qiagen PCR cleanup kit.
 +
Determined concentrations by nanodrop.  KanA/B': 101.1ng/uL KanB/A':89.6ng/uL
 +
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=18-05-2010=
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Prepared DH5&alpha; E.Coli competent cells using the Inoue Method.
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Transformed DH5&alpha; cells with pSB1C3-J04450 and grew overnight at 37^o^C on Chloramphenicol plates
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 +
=19-05-2010=
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From the transformation of DH5&alpha; 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.
 +
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=20-05-2010=
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Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5&alpha; cells with pSB1C3-J04450 from [[#19-05-2010|19-05-2010]].  Took a 1uL sample of the Miniprep solutions and digested with NotI at 37^o^C for 1 hour.
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Digestion Recipe:
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:1uL Miniprep (between 153.2 ng/ul and 302.7ng/ul determined by nanodrop)
 +
:1uL NotI
 +
:1uL 10X ReACT 3
 +
:7uL MilliQ
 +
 +
<!-- Image of Gel-->
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=27-05-2010=
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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^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 DH5&alpha; 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
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 +
=28-05-2010=
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=June 3, 2009=
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We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary
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#Plasmid transformed = pSB1AC3 (TEST)  
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#*Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.
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=June 5, 2009=
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=30-05-2010=
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#Tet plates made
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#:Recipe for 200 ml (approx. 10 plates):
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#::2.2 g agar in 200 ml fresh LB
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#::Note: do not re-autoclave LB, it will caramelize!
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#:Recipe for 200 ml LB:
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#::a) 1 g yeast extract
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#::b) 2 g peptotryptone
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#::c) 2 g NaCl
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#::d) 200 ml water
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#Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
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#Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
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#Swirl and poured into prepared, labeled plates
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#*Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
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#Inverted and put in 37 degree incubator to dry
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=June 8, 2009=
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From the transformation of DH5&alpha; 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.
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#Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)
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#Bacterial liquid culture placed in shaker at 10:51 a.m.
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=June 9, 2009=
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=31-05-2010=
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#Digested miniprepped gel with EcoRI and SpeI
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#Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
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#DNA Ladder made - 6 microlitres of stock used
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Revision as of 21:52, 31 May 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:

  1. 5 min-94^o^C
  2. 45 sec-94^o^C
  3. 1 min-60^o^C
  4. 1 min-72^o^C
  5. Repeat 2 through 4 35 times
  6. 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

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