Team:UCLA/Notebook/Library

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<br><br>
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<h1>Generating the Mtd Library</h1>
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<p>Using PCR, we made several modifications to the <i>mtd</i> gene in order to generate our diverse library of <i>mtd</i> variants in a mRNA display-compatible format. Two sequential PCRs were used to generate the library. Primers and protocols are listed below.</p>
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<table id="tfhover" class="tftable" border="1">
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<tr><th>Primer Number</th><th>Primer Sequence</th>
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<tr><td>P10 Forward Primer</td><td>TATTGTAATACGACTCACTATAGGGCATTAGGAGGccaaCATTGCCACCatgagtaccgcagtccagttccg
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</td>
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<tr><td>P11 Primer C</td><td>gccggaCTGCAGCTTATCGTCGTCATCCTTGTAATCctcaagaatcaggtggtcacagacGCCGCGCGCCCC
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<tr><td>P12 Primer B</td><td>cagacGCCGCGCGCCCCGANGNNCGCGNNCGAGNNCGACGGCCCGNNGNNCCAGNNcgcagcgcgagaacccgag
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</td>
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<tr><td>P13 Primer A</td><td>cgcagcgcgagaacccgagNNcgacgtgNNgNNccagNNgccgccgaatagcgcagcagc
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</td>
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<tr><td>P14 Splint</td><td>TTTTTTTTTTTTgccggaCTGCAG
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</td>
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[[File:Mutagenic_primer_design.PNG|thumb|right|Oligomer design for generation of Mtd mutagenic islands]]
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<br>
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<h4>Generation of <i>mtd</i> library containing first mutagenic island</h4>
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<table id="tfhover" class="tftable" border="1">
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<tr><th>Reagent</th><th>Volume (uL)</th>
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<tr><td>Water</td><td>11.8</td>
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<tr><td>Phusion Buffer HF</td><td>4
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</td>
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<tr><td>DMSO</td><td>0.6
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</td>
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<tr><td>dNTP (10 mM)</td><td>0.4</td>
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<tr><td>Bordetella Phage Genomic Template</td><td>1
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</td>
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<tr><td>P10 Forward Primer (10 uM)</td><td>1</td>
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<tr><td>P13 Primer A (10 uM)</td><td>1</td>
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<tr><td>Phusion DNA Polymerase</td><td>0.2</td>
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<tr><th># Cycles</th><th>Temperature (&#176;C)</th><th>Time</th></tr>
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<tr><td>1</td><td>98</td><td>0:30</td></tr>
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<tr><td>30</td><td>98<br>65<br>72</td><td>0:10<br>0:20<br>0:25</td></tr>
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<tr><td>1</td><td>72</td><td>8:00</td></tr>
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<tr><td>1</td><td>4</td><td>--</td></tr>
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<br>
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<h4>Generation of full <i>mtd</i> library containing second mutagenic island, FLAG tag, and splint attachment site</h4>
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<p>In this bridging PCR, a higher concentration of the outermost primer was used relative to the middle primer in order to maximize the chance that all products are full-length (both the middle and outermost primers bind). Equal concentrations of primers resulted in a broad ladder smear.
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<tr><th>Reagent</th><th>Volume (uL)</th>
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<tr><td>Water</td><td>3.9</td>
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<tr><td>KOD 2X Xtreme Buffer</td><td>10
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</td>
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<tr><td>dNTPs (10 mM)</td><td>4
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</td>
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<tr><td>P10 Forward Primer (10 uM)</td><td>0.6</td>
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<tr><td>P12 Primer B (10 uM)</td><td>0.1
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</td>
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<tr><td>P16 Primer A (10 uM)</td><td>0.5</td>
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<tr><td><i>mtd</i> PCR Product</td><td>0.5 (20 ng)</td>
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<tr><td>KOD Xtreme Hot Start Polymerase</td><td>0.4</td>
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<tr><th># Cycles</th><th>Temperature (&#176;C)</th><th>Time</th></tr>
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<tr><td>1</td><td>94</td><td>2:00</td></tr>
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<tr><td>35</td><td>98<br>64<br>68</td><td>0:10<br>0:30<br>1:20</td></tr>
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<tr><td>1</td><td>4</td><td>--</td></tr>
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Revision as of 23:11, 27 September 2013




Generating the Mtd Library

Using PCR, we made several modifications to the mtd gene in order to generate our diverse library of mtd variants in a mRNA display-compatible format. Two sequential PCRs were used to generate the library. Primers and protocols are listed below.

Primer NumberPrimer Sequence
P10 Forward PrimerTATTGTAATACGACTCACTATAGGGCATTAGGAGGccaaCATTGCCACCatgagtaccgcagtccagttccg
P11 Primer CgccggaCTGCAGCTTATCGTCGTCATCCTTGTAATCctcaagaatcaggtggtcacagacGCCGCGCGCCCC
P12 Primer BcagacGCCGCGCGCCCCGANGNNCGCGNNCGAGNNCGACGGCCCGNNGNNCCAGNNcgcagcgcgagaacccgag
P13 Primer AcgcagcgcgagaacccgagNNcgacgtgNNgNNccagNNgccgccgaatagcgcagcagc
P14 SplintTTTTTTTTTTTTgccggaCTGCAG

Oligomer design for generation of Mtd mutagenic islands


Generation of mtd library containing first mutagenic island


ReagentVolume (uL)
Water11.8
Phusion Buffer HF4
DMSO0.6
dNTP (10 mM)0.4
Bordetella Phage Genomic Template1
P10 Forward Primer (10 uM)1
P13 Primer A (10 uM)1
Phusion DNA Polymerase0.2


# CyclesTemperature (°C)Time
1980:30
3098
65
72		0:10
0:20
0:25
1728:00
14--


Generation of full mtd library containing second mutagenic island, FLAG tag, and splint attachment site

In this bridging PCR, a higher concentration of the outermost primer was used relative to the middle primer in order to maximize the chance that all products are full-length (both the middle and outermost primers bind). Equal concentrations of primers resulted in a broad ladder smear.

ReagentVolume (uL)
Water3.9
KOD 2X Xtreme Buffer10
dNTPs (10 mM)4
P10 Forward Primer (10 uM)0.6
P12 Primer B (10 uM)0.1
P16 Primer A (10 uM)0.5
mtd PCR Product0.5 (20 ng)
KOD Xtreme Hot Start Polymerase0.4

# CyclesTemperature (°C)Time
1942:00
3598
64
68		0:10
0:30
1:20
14--

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