Team:Alberta/Building Parts

From 2010.igem.org

(Difference between revisions)
Line 6: Line 6:
   
   
Recipe:  
Recipe:  
-
:1uL p1003 (approx. 1ng)
+
:1μL p1003 (approx. 1ng)
-
:2.5uL prA_p1003+
+
:2.5μL prA_p1003+
-
:2.5uL prB'_p1003-
+
:2.5μL prB'_p1003-
-
:5uL 10X PCR buffer
+
:5μL 10X PCR buffer
-
:1uL 10uM dNTPs
+
:1μL 10uM dNTPs
-
:2uL 50uM MgCl<sub>2</sub>  
+
:2&mu;L 50uM MgCl<sub>2</sub>  
-
:0.5uL Taq polymerase
+
:0.5&mu;L Taq polymerase
-
:35.5uL MilliQ H<sub>2</sub>O
+
:35.5&mu;L MilliQ H<sub>2</sub>O
Program:
Program:
Line 23: Line 23:
# 5 min-72<sup>o</sup>C
# 5 min-72<sup>o</sup>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. 5&mu;L of each PCR reaction, 1&mu;L of 10X loading dye and 4&mu;L MilliQ water in a 1% agarose gel -->
=11-05-2010=
=11-05-2010=
PCR purification of Kanamycin Resistant fragments created [[#10-05-2010|10-05-2010]] with Qiagen PCR cleanup kit.
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
+
Determined concentrations by nanodrop.  KanA/B': 101.1ng/&mu;L KanB/A':89.6ng/&mu;L
=18-05-2010=
=18-05-2010=
Line 41: Line 41:
=20-05-2010=
=20-05-2010=
-
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<sup>o</sup>C for 1 hour.  
+
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 1&mu;L sample of the Miniprep solutions and digested with NotI at 37<sup>o</sup>C for 1 hour.  
Digestion Recipe:
Digestion Recipe:
-
:1uL Miniprep (between 153.2 ng/ul and 302.7ng/ul determined by nanodrop)
+
:1&mu;L Miniprep (between 153.2 ng/&mu;l and 302.7ng/&mu;l determined by nanodrop)
-
:1uL NotI
+
:1&mu;L NotI
-
:1uL 10X ReACT 3
+
:1&mu;L 10X ReACT 3
-
:7uL MilliQ
+
:7&mu;L MilliQ
<!-- Image of Gel-->
<!-- Image of Gel-->
Line 53: Line 53:
=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 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.
+
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 15&mu;L to room temperature for 2 hours.  Transformed 100&mu;L of DH5&alpha; cells with 5&mu;L of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.
Digestion Recipe:
Digestion Recipe:
-
:1uL Miniprep (302.7ng/ul determined by nanodrop)
+
:1&mu;L Miniprep (302.7ng/&mu;l determined by nanodrop)
-
:2uL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/uL)
+
:2&mu;L either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/&mu;L)
-
:1uL NotI
+
:1&mu;L NotI
-
:1uL 10X ReACT 3  
+
:1&mu;L 10X ReACT 3  
-
:5uL MilliQ
+
:5&mu;L MilliQ
Ligation Recipe:
Ligation Recipe:
-
:10uL of Digest solution
+
:10&mu;L of Digest solution
-
:1uL T4 DNA ligase  
+
:1&mu;L T4 DNA ligase  
-
:6uL 5X Buffer
+
:6&mu;L 5X Buffer
-
:13uL MilliQ H<sub>2</sub>O
+
:13&mu;L MilliQ H<sub>2</sub>O
=28-05-2010=
=28-05-2010=

Revision as of 19:30, 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:

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:

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

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.

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 and at Room Temperature for 3 hours.