Team:UIUC-Illinois/Notebook

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         <div class="title">Plasmid Integration Protocol (for CRIM plasmids)</div>
         <div class="title">Plasmid Integration Protocol (for CRIM plasmids)</div>
-
 
-
        <div class="desc"><img src="https://static.igem.org/mediawiki/2011/5/52/Illinois_igem_team_s.jpg" alt="Illinois iGEM Team" /></div>
 
         <div class="desc">1. Prepare electrocompetent cells that have been transformed with the helper plasmid (pAH57) and that have been grown in 5 mL SOB to an OD of 600nm of ca. 0.6 with ampicillin antibiotic at 30 degC. </div>
         <div class="desc">1. Prepare electrocompetent cells that have been transformed with the helper plasmid (pAH57) and that have been grown in 5 mL SOB to an OD of 600nm of ca. 0.6 with ampicillin antibiotic at 30 degC. </div>
Line 47: Line 45:
         <div class="desc"><img src="https://static.igem.org/mediawiki/2011/9/9e/Uiuc_notebook_1.jpg" /></div>
         <div class="desc"><img src="https://static.igem.org/mediawiki/2011/9/9e/Uiuc_notebook_1.jpg" /></div>
-
         <div class="desc"></div>
+
         <div class="desc">The following primers were then designed in order to amplify the highlighted (red)section below.  The primers contain overhangs such that subsequent SpeI digestion and an intramolecular ligation of the pcr product will produce a biobrick cloning site, which replaces the original MCS and lacZ ORF in the pAH125 diagram.</div>
-
         <div class="desc"></div>
+
         <div class="desc"><img src="https://static.igem.org/mediawiki/2011/6/6a/Uiuc_notebook_2.jpg" /></div>
-
         <div class="desc"></div>
+
         <div class="desc">Primers:</div>
-
         <div class="desc"></div>
+
         <div class="desc">ATCG  <font color="red">T ACTAGT A GCGGCCG CTGCAG</font> CAGTGAATTAATGGCGATGACGC</div>
 +
 
 +
        <div class="desc">Tm = 68 C</div>
 +
 
 +
        <div class="desc">GC = 48%</div>
 +
 
 +
        <div class="desc">ATCG ACTAGTA <font color="green">CTCTAGAAGCGGCCGCGAATTC</font>  GCATGCAAGCTTGGCACTGG</div>
 +
 
 +
        <div class="desc">Tm = 64 C</div>
 +
 
 +
        <div class="desc">GC = 60%</div>
 +
 
 +
        <br><br>
 +
 
 +
        <div class="desc">The following PCR rxn was then set up and run with the subsequent parameters:</div>
 +
        <div class="desc">0.3 uL pAH125</div>
 +
        <div class="desc">2uL primer 1 (100ng/uL)</div>
 +
        <div class="desc">2uL primer2 (100ng/uL)</div>
 +
        <div class="desc">5uL 10X Pfu buffer (Agilent)</div>
 +
        <div class="desc">2.5uL 10mM dNTP mix</div>
 +
        <div class="desc">1uL Turbo Polymerase (Agilent)</div>
 +
        <div class="desc">37uL ddH2O</div>
 +
 
 +
        <br><br>
 +
 
 +
        <div class="desc">95C 5 min</div>
 +
        <div class="desc">95C 30sec</div>
 +
        <div class="desc">59C 30 sec</div>
 +
        <div class="desc">72C 3 min</div>
 +
        <div class="desc">Cycle 30X</div>
 +
        <div class="desc">72C 5 min</div>
 +
        <div class="desc">14C forever</div>
 +
 
 +
        <br><br>
 +
 
 +
        <div class="desc">The pcr rxn was then  purified using a Qiagen pcr purification kit.  Resulting concentration was 99ng/uL.</div>
 +
        <div class="desc">500ng of the reaction was then digested SpeI with the following reaction set up:</div>
 +
        <div class="desc">0.5uL pcr product</div>
 +
        <div class="desc">5uL NEB Buffer 2</div>
 +
        <div class="desc">1uL 100X BSA</div>
 +
        <div class="desc">1uL SpeI (from NEB)</div>
 +
        <div class="desc">42.5uL ddH2O</div>
 +
 
 +
        <br><br>
 +
 
 +
        <div class="desc">Digest ran 1 hour 37C and was followed by an 80C heat inactivation.</div>
 +
 
 +
 
 +
        <br><br>
 +
 
 +
 +
        <div class="desc">2uL of the digestion reaction was then used in a 20 uL volume ligation reaction.  Set up was as follows:</div>
 +
 
 +
 
 +
        <br><br>
 +
 
 +
 +
        <div class="desc">2uL SpeI digest</div>
 +
        <div class="desc">2uL 10X Promega T4 ligase buffer</div>
 +
        <div class="desc">1uL Promega T4 ligase</div>
 +
        <div class="desc">15uL ddH2O</div>
 +
 
 +
 
 +
        <br><br>
 +
 
 +
 +
        <div class="desc">Ligation reaction ran at for 8 hours at 15C.</div>
 +
 
 +
 
 +
        <br><br>
 +
 
 +
 +
        <div class="desc">3uL of the ligation reaction was then transformed into two strains via heat shock as described in our protocol download.  The first strain was a normal pir- DH5alpha E. coli strain and the second was a E. coli K-12 pir-116 strain.  The latter should allow replication of the plasmid while the first should not.  The transformations were plated on LB kan 30ug/mL.  Plates were incubated 18 hours at 37C.  The following pictures show that robust colonies resulted on the pir-116 transformant plate but not on the pir- DH5alpha plate.</div>
 +
 
 +
 
 +
        <br><br>
 +
 
 +
 +
        <div class="desc"><img src="https://static.igem.org/mediawiki/2011/d/da/Uiuc_notebook_3.jpg" /></div>
 +
 
 +
        <div class="desc">(above) pir-116 transformations, (below) DH5alpha pir- transformation</div>
 +
 
 +
        <div class="desc"><img src="https://static.igem.org/mediawiki/2011/5/5e/Uiuc_notebook_4.jpg" /></div>
 +
 
 +
        <div class="desc">Canidate colonies were then picked off the pir-116 plate and grown up in 6mL LB kanamycin 30ug/mL.  The resulting cultures were miniprepped and the products digested EcoRI, PstI.  The resulting restriction profile was expected to yield a 2.4 kb band.  The four canidates were run on a gel (see the lower left quadrant of the below picture:</div>
 +
 
 +
        <div class="desc"><img src="https://static.igem.org/mediawiki/2011/d/dd/Uiuc_notebook_5.jpg" /></div>
 +
 
 +
        <div class="desc">Three out of the four candidates were a match, the top most candidate was sent in as K617000.  It has showed the correct restriction profile of 2.4kb when digested with EcoRI and PstI and as been shown to replicate in pir-116 but not DH5alpha pir-.  A final gel of K617000 digested EcoRI, PstI is shown below, highlighted in red.</div>
 +
 
 +
        <div class="desc"><img src="https://static.igem.org/mediawiki/2011/e/e9/Uiuc_notebook_6.jpg" /></div>
 +
 
 +
        <div class="desc">The restriction profiles of submitted parts K617003 and 004 are also shown on the same gel above (EcoRI, PstI digested).</div>
 +
 
 +
        <div class="title">Plasmid Integration Protocol (for CRIM plasmids)</div>
 +
        <div class="desc">K617003 and K617004 Creation</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">Set up pcr reactions using the pAH125 template miniprep that was also used for the creation of the K617000 plasmid.  Concentration is 30ng/uL</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">Lambda attP P’OP pcr (K617004)</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">Primer 1</div>
 +
        <div class="desc">ATCG GAATTC GCGGCCGC T TCTAGA G  CCATGGCATCACAGTATCGTG</div>
 +
        <div class="desc">Tm =  64 C (eq)</div>
 +
        <div class="desc">Primer 2</div>
 +
        <div class="desc">ATCG CTGCAGCGGCCGCTACTAGTA AGCTTTGCACTGGATTGCGAG</div>
 +
        <div class="desc">Tm = 64 C (eq)</div>
 +
        <div class="desc">GC=52% </div>
 +
 
 +
        <br><br>
 +
        <div class="desc">0.3uL pAH125</div>
 +
        <div class="desc">2uL primer 1 (100ng/uL)</div>
 +
        <div class="desc">2uL primer2 (100ng/uL)</div>
 +
        <div class="desc">5uL 10X Pfu Buffer</div>
 +
        <div class="desc">2.5uL 10mM dNTP mix</div>
 +
        <div class="desc">1uL Turbo Polymerase</div>
 +
        <div class="desc">37uL ddH2O</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">95C 5 min</div>
 +
        <div class="desc">95C 30 sec</div>
 +
        <div class="desc">59C 30 sec</div>
 +
        <div class="desc">72C 1 min</div>
 +
        <div class="desc">Cycle 30X</div>
 +
        <div class="desc">72C 5 min</div>
 +
        <div class="desc">4C forever</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">R6K origin pcr (K617003)</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">Primer 1’</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">ATCG  GAATTC GCGGCCGC T TCTAGA G CCATGTCAGCCGTTAAGTGTTCC</div>
 +
        <div class="desc">Tm(eq)= 70 C</div>
 +
        <div class="desc">GC = 52%</div>
 +
 
 +
 
 +
        <br><br>
 +
        <div class="desc">Primer2’</div>
 +
        <div class="desc">ATCG CTGCAGCGGCCGCTACTAGTA GATCTGAAGATCAGCAGTTCAACC</div>
 +
        <div class="desc">Tm(eq)= 70 C</div>
 +
        <div class="desc">GC = 46%</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">0.3uL pAH125</div>
 +
        <div class="desc">2uL primer 1’ (100ng/uL)</div>
 +
        <div class="desc">2uL primer2’ (100ng/uL)</div>
 +
        <div class="desc">5uL 10X Pfu Buffer</div></div>
 +
        <div class="desc">2.5uL 10mM dNTP mix</div>
 +
        <div class="desc">1uL Turbo Polymerase</div>
 +
        <div class="desc">37uL ddH2O</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">95C 5 min</div>
 +
        <div class="desc">95C 30 sec</div>
 +
        <div class="desc">65C 30 sec</div>
 +
        <div class="desc">72C 1 min</div>
 +
        <div class="desc">Cycle 30X</div>
 +
        <div class="desc">72C 5 min</div>
 +
        <div class="desc">4C forever</div>
 +
 
 +
        <br><br>
 +
        <div class="desc">Both pcr reactions were pcr purified using Qiagen pcr purification kits.  Both concentrations were approximately 90 ng/uL. </div>
 +
 
 +
        <br><br>
 +
        <div class="desc">Products were then digested EcoRI PstI and assembled with pSB1C3 also cut EcoRI PstI to release an RFP generator.  Ligation reactions were performed as previously described (between pSB1C3 and the digested pcr fragments).  LB Chloramphenicol 25ug/mL were used for selection as well as red white selection as seen in pictures below.  </div>
 +
 
 +
        <div class="desc"><img src="https://static.igem.org/mediawiki/2011/a/af/Uiuc_notebook_7.jpg" /></div>
 +
 
 +
        <div class="desc">The three section gel previsouly shown confirms the correct canidates were picted for K617003 and 004.</div>
       </div>
       </div>
     </div>
     </div>
 +
 +
<div class="desc"><a href="url">http://openwetware.org/wiki/IGEM:UIUC-Illinois/2011/Notebook/UIUC_Illinois_iGEM_2011</a></div>
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Latest revision as of 04:01, 29 September 2011

University of Illinois iGEM Team
About the Team
Team Navigation
Notebook
OpenWetWare Notebook

Who We Are
Amanda Chang
"A watched gel never runs"
Plasmid Integration Protocol (for CRIM plasmids)
1. Prepare electrocompetent cells that have been transformed with the helper plasmid (pAH57) and that have been grown in 5 mL SOB to an OD of 600nm of ca. 0.6 with ampicillin antibiotic at 30 degC.
2. Perform an electroporation transformation with the BioBrick Compatible Lambda Chromosomal Insertion Plasmid making sure to resuspend the cells in 1 mL of SOC (without ampicillin). Incubate at 37 degC for 1 hour, and then 42 degC for 30 minutes. Spread plate onto a selective agar plate and incubate at 37 degC.
K617000 Creation
Day 1
The CRIM pAH125 vector was obtained in a pir+ strain. The culture was streaked from the -80C onto a LB kanamycin 30ug/mL plate. Plate was incubated 37C for 18 hours. White colonies resulted.
A single colony was picked from the previous plate and was used to inoculate 6mL of LB kanamycin 30ug/mL. The culture was then incubated 37C on a tube turner for 16 hours. A turbid yellow-white culture resulted.
5mL of the culture was pelleted and miniprepped using the Promega mini-prep kit and protocol described in our protocol download. The resulting concentration was 30ng/uL.
The plasmid map of pAH125 is shown below:
The following primers were then designed in order to amplify the highlighted (red)section below. The primers contain overhangs such that subsequent SpeI digestion and an intramolecular ligation of the pcr product will produce a biobrick cloning site, which replaces the original MCS and lacZ ORF in the pAH125 diagram.
Primers:
ATCG T ACTAGT A GCGGCCG CTGCAG CAGTGAATTAATGGCGATGACGC
Tm = 68 C
GC = 48%
ATCG ACTAGTA CTCTAGAAGCGGCCGCGAATTC GCATGCAAGCTTGGCACTGG
Tm = 64 C
GC = 60%


The following PCR rxn was then set up and run with the subsequent parameters:
0.3 uL pAH125
2uL primer 1 (100ng/uL)
2uL primer2 (100ng/uL)
5uL 10X Pfu buffer (Agilent)
2.5uL 10mM dNTP mix
1uL Turbo Polymerase (Agilent)
37uL ddH2O


95C 5 min
95C 30sec
59C 30 sec
72C 3 min
Cycle 30X
72C 5 min
14C forever


The pcr rxn was then purified using a Qiagen pcr purification kit. Resulting concentration was 99ng/uL.
500ng of the reaction was then digested SpeI with the following reaction set up:
0.5uL pcr product
5uL NEB Buffer 2
1uL 100X BSA
1uL SpeI (from NEB)
42.5uL ddH2O


Digest ran 1 hour 37C and was followed by an 80C heat inactivation.


2uL of the digestion reaction was then used in a 20 uL volume ligation reaction. Set up was as follows:


2uL SpeI digest
2uL 10X Promega T4 ligase buffer
1uL Promega T4 ligase
15uL ddH2O


Ligation reaction ran at for 8 hours at 15C.


3uL of the ligation reaction was then transformed into two strains via heat shock as described in our protocol download. The first strain was a normal pir- DH5alpha E. coli strain and the second was a E. coli K-12 pir-116 strain. The latter should allow replication of the plasmid while the first should not. The transformations were plated on LB kan 30ug/mL. Plates were incubated 18 hours at 37C. The following pictures show that robust colonies resulted on the pir-116 transformant plate but not on the pir- DH5alpha plate.


(above) pir-116 transformations, (below) DH5alpha pir- transformation
Canidate colonies were then picked off the pir-116 plate and grown up in 6mL LB kanamycin 30ug/mL. The resulting cultures were miniprepped and the products digested EcoRI, PstI. The resulting restriction profile was expected to yield a 2.4 kb band. The four canidates were run on a gel (see the lower left quadrant of the below picture:
Three out of the four candidates were a match, the top most candidate was sent in as K617000. It has showed the correct restriction profile of 2.4kb when digested with EcoRI and PstI and as been shown to replicate in pir-116 but not DH5alpha pir-. A final gel of K617000 digested EcoRI, PstI is shown below, highlighted in red.
The restriction profiles of submitted parts K617003 and 004 are also shown on the same gel above (EcoRI, PstI digested).
Plasmid Integration Protocol (for CRIM plasmids)
K617003 and K617004 Creation


Set up pcr reactions using the pAH125 template miniprep that was also used for the creation of the K617000 plasmid. Concentration is 30ng/uL


Lambda attP P’OP pcr (K617004)


Primer 1
ATCG GAATTC GCGGCCGC T TCTAGA G CCATGGCATCACAGTATCGTG
Tm = 64 C (eq)
Primer 2
ATCG CTGCAGCGGCCGCTACTAGTA AGCTTTGCACTGGATTGCGAG
Tm = 64 C (eq)
GC=52%


0.3uL pAH125
2uL primer 1 (100ng/uL)
2uL primer2 (100ng/uL)
5uL 10X Pfu Buffer
2.5uL 10mM dNTP mix
1uL Turbo Polymerase
37uL ddH2O


95C 5 min
95C 30 sec
59C 30 sec
72C 1 min
Cycle 30X
72C 5 min
4C forever


R6K origin pcr (K617003)


Primer 1’


ATCG GAATTC GCGGCCGC T TCTAGA G CCATGTCAGCCGTTAAGTGTTCC
Tm(eq)= 70 C
GC = 52%


Primer2’
ATCG CTGCAGCGGCCGCTACTAGTA GATCTGAAGATCAGCAGTTCAACC
Tm(eq)= 70 C
GC = 46%


0.3uL pAH125
2uL primer 1’ (100ng/uL)
2uL primer2’ (100ng/uL)
5uL 10X Pfu Buffer
2.5uL 10mM dNTP mix
1uL Turbo Polymerase
37uL ddH2O


95C 5 min
95C 30 sec
65C 30 sec
72C 1 min
Cycle 30X
72C 5 min
4C forever


Both pcr reactions were pcr purified using Qiagen pcr purification kits. Both concentrations were approximately 90 ng/uL.


Products were then digested EcoRI PstI and assembled with pSB1C3 also cut EcoRI PstI to release an RFP generator. Ligation reactions were performed as previously described (between pSB1C3 and the digested pcr fragments). LB Chloramphenicol 25ug/mL were used for selection as well as red white selection as seen in pictures below.
The three section gel previsouly shown confirms the correct canidates were picted for K617003 and 004.
http://openwetware.org/wiki/IGEM:UIUC-Illinois/2011/Notebook/UIUC_Illinois_iGEM_2011

Retrieved from "http://2011.igem.org/Team:UIUC-Illinois/Notebook"