Team:Alberta/Building Parts

From 2010.igem.org

(Difference between revisions)
(→10-05-2010)
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:5uL 10X PCR buffer
:5uL 10X PCR buffer
:1uL 10uM dNTPs
:1uL 10uM dNTPs
-
:2uL 50uM MgCl~2~
+
:2uL 50uM MgCl<sub>2</sub>
:0.5uL Taq polymerase
:0.5uL Taq polymerase
-
:35.5uL MilliQ H~2~O
+
:35.5uL MilliQ H<sub>2</sub>O
Program:
Program:
-
# 5 min-94^o^C
+
# 5 min-94<sup>o</sup>C
-
# 45 sec-94^o^C
+
# 45 sec-94<sup>o</sup>C
-
# 1 min-60^o^C
+
# 1 min-60<sup>o</sup>C
-
# 1 min-72^o^C
+
# 1 min-72<sup>o</sup>C
# Repeat 2 through 4 35 times
# Repeat 2 through 4 35 times
-
# 5 min-72^o^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. 5uL of each PCR reaction, 1uL of 10X loading dye and 4uL MilliQ water in a 1% agarose gel -->
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Prepared DH5&alpha; E.Coli competent cells using the Inoue Method.  
Prepared DH5&alpha; E.Coli competent cells using the Inoue Method.  
-
Transformed DH5&alpha; cells with pSB1C3-J04450 and grew overnight at 37^o^C on Chloramphenicol plates
+
Transformed DH5&alpha; cells with pSB1C3-J04450 and grew overnight at 37<sup>o</sup>C on Chloramphenicol plates
=19-05-2010=
=19-05-2010=
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=20-05-2010=
=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.  
+
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.  
Digestion Recipe:
Digestion Recipe:
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^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.
+
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.
Digestion Recipe:
Digestion Recipe:
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:1uL T4 DNA ligase  
:1uL T4 DNA ligase  
:6uL 5X Buffer
:6uL 5X Buffer
-
:13uL MilliQ H~2~O
+
:13uL MilliQ H<sub>2</sub>O
=28-05-2010=
=28-05-2010=
Line 74: Line 74:
=30-05-2010=
=30-05-2010=
-
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.
+
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 overnight at 37<sup>o</sup>C.
=31-05-2010=
=31-05-2010=
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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.
-
Performed a restriction digest of an aliquot of the pSB1C3-KanA/B' and pSB1C3-KanB/A' minipreps.  Digested with XbaI at 37^o^C for one hour and then with EcoRI at 37^o^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 DATE, digested with BsaI at 37^o^C for 1.5hours, heat inactivated at 65^o^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^o^C and at Room Temperature for 3 hours.
+
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 Room Temperature for 3 hours.

Revision as of 19:24, 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:

1uL p1003 (approx. 1ng)
2.5uL prA_p1003+
2.5uL prB'_p1003-
5uL 10X PCR buffer
1uL 10uM dNTPs
2uL 50uM MgCl2
0.5uL Taq polymerase
35.5uL 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/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 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 1uL sample of the Miniprep solutions and digested with NotI at 37oC 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 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 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 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.

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