Team:UCLA/Notebook/Biobrick

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Latest revision as of 23:28, 27 September 2013

1 Making the Mtd Biobrick

Making the Mtd Biobrick

To create this biobrick, we utilized a combination of splicing overlap extension PCR and Gibson Assembly to extract a sequence from the BPP-1 phage's genomic DNA that would be free of the standard biobrick restriction enzyme sites. The protocols for those methods are listed below:

PCR to generate fragments of mtd

In this step, four separate fragments of the mtd gene are created via PCR. This step is necessary to eliminate illegal internal biobrick sites (EcoRI, PstI, NotI) and to add necessary biobrick sites to the 5' and 3' ends.

Fragment	Forward Primer	Reverse Primer
1	CCTTGAATTCGCGGCCGCATCTAGAATGAGTACCGCAGTCCAGTTCCG	GCCTGCGCTGCCGCGTTGCTTCC
2	GGAAGCAACGCGGCAGCGCAGGC	CCAGCGCCTGGAACTCGTTGTAGTTGGGCAGG
3	CCTGCCCAACTACAACGAGTTCCAGGCGCTGG	CCGATCCGCGGCCTCCAGTGTTGG
4	CCAACACTGGAGGCCGCGGATCGG	AAGGCTGCAGCGGCCGCTACTAGTCTCAAGAATCAGG

The 20 µL PCR mix for each fragment is as follows:

Reagent	Volume
mtd genomic template	1.0 µL (4.5 ng total)
10 µM forward primer	1.0 µL
10 µM reverse primer	1.0 µL
10 mM dNTPs	0.4 µL
Buffer HF	4.0 µL
Phusion Polymerase	0.2 µL
ddH₂O	12.4 µL

The thermocycler program for each reaction is as follows:

# Cycles	Temperature (°C)	Time
1	98	0:30
30	98 Variable (70 for fragment 1, 69 for fragment 2, 69 for fragment 3, 72 for fragment 3) 72	0:10 0:20 0:15
1	72	5:00
1	4	--

The PCR product is then resolved on a 1% agarose gel and excised and column-extracted.

Splicing Overlap-Extension (SOE) PCR to connect fragments 3 and 4

The 20 µL reaction mix is as follows:

Reagent	Volume or Mass
Fragment 3	20 ng total
Fragment 4	20 ng total
10 µM forward primer	1.0 µL
10 µM reverse primer	1.0 µL
10 mM dNTPs	0.4 µL
Buffer HF	4.0 µL
Phusion Polymerase	0.2 µL
ddH₂O	fill to 20 µL

The thermocycler program for this step is as follows:

# Cycles	Temperature (°C)	Time
1	98	0:30
30	98 72 72	0:10 0:20 0:15
1	72	5:00
1	4	--

Gibson Assembly of Fragments 1,2, and 3-4

The mix for the Gibson Assembly is as follows:

Reagent	Concentration or Volume
Fragment 1	0.2 pM
Fragment 2	0.2 pM
Fragment 3-4	0.2 pM
Assembly Master Mix (from New England Biolabs)	fill to 5.0 µL

The mix is then incubated at 50 °C for 1 hour.

PCR amplification of mtd

The mix for the amplification is as follows:

Reagent	Volume or Mass
mtd	20 ng
10 µM forward primer (CCTTGAATTCGCGGCCGCATCTAGAATGAGTACCGCAGTCCAGTTCCG )	0.6 µL
10 µM reverse primer (AAGGCTGCAGCGGCCGCTACTAGTCTCAAGAATCAGG )	0.6 µL
10 mM dNTPs	0.4 µL
2x xtreme buffer	10 µL
KOD xtreme hot start polymerase	0.4 µL
ddH₂O	fill to 20 µL

The thermocycler program for the amplification is as follows:

# Cycles	Temperature (°C)	Time
1	94	2:00
35	98 70 68	0:10 0:30 1:20
1	10	--

Retrieved from "http://2013.igem.org/Team:UCLA/Notebook/Biobrick"

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 	<li><a href="/Team:UCLA/Modeling">MODELING</a></li>
 	<li><a href="/Team:UCLA/HumanPractices">HUMAN PRACTICES</a></li>
-	<li><div id="spec"><a href="/Team:UCLA/Notebook"><font color="black">NOTEBOOK</font></a></div></li>
+	<li><div id="spec"><a href="/Team:UCLA/Notebook/Biobrick"><font color="black">NOTEBOOK</font></a></div></li>
 	<li><a href="/Team:UCLA/Safety">SAFETY</a></li>
          <li><a href="/Team:UCLA/Attributions">ATTRIBUTIONS</a></li>
@@ Line 54: / Line 54: @@
 <html>
 <ul id="subnav">
-	<li style="margin-left: 12px;"><a href = "https://2013.igem.org/Team:UCLA/Notebook/Overview">Overview</a></li>
-        <li id="current"><a href="https://2013.igem.org/Team:UCLA/Notebook/Biobrick">Mtd Biobrick Construction</a></li>
+        <li style="margin-left: 12px;" id="current"><a href="https://2013.igem.org/Team:UCLA/Notebook/Biobrick">Mtd Biobrick Construction</a></li>
 	<li><a href="/Team:UCLA/Notebook/Library">mRNA Display Library Generation</a></li>
 	<li><a href="/Team:UCLA/Notebook/mRNAdisplay">mRNA Display</a></li>