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| | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
| | ==December 5, 2012== | | ==December 5, 2012== |
| - | ==11/21/12==
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| - |
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| | '''Results from last week's ligation''' | | '''Results from last week's ligation''' |
| | * None of the colonies on any plates grew | | * None of the colonies on any plates grew |
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| | * Clean up PCR products using the Zymo Clean and Concentrator kit | | * Clean up PCR products using the Zymo Clean and Concentrator kit |
| | * Be sure to elute using 15 μL dH<sub>2</sub>O | | * Be sure to elute using 15 μL dH<sub>2</sub>O |
| - |
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| - | ----
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| - |
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| - | *'''[[User:Karmella Haynes|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation
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| - |
| |
| - | '''Assemblies'''
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| - | # NEB T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
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| - | # NEB T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
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| - | # NEB T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp
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| - | # Roche T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
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| - | # Roche T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
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| - | # Roche T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp
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| - |
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| - | * Digests:
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| - | ** In a 30 μL reaction, cut H2B with XbaI and SpeI
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| - | ** In a 30 μL reaction, cut LOV with XbaI and SpeI
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| - |
| |
| - | {| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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| - | |-
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| - | | Reagent || H2B PCR || LOV PCR
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| - | |-
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| - | | DNA || 15.0 || 15.0
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| - | |-
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| - | | XbaI || 1.0|| 1.0
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| - | |-
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| - | | SpeI || 1.0 || 1.0
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| - | |-
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| - | | 10x buffer || 3.0 || 3.0
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| - | |-
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| - | | dH<sub>2</sub>O || 10.0 || 10.0
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| - | |-
| |
| - | | Total vol. || 30. 0 || 30.0
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| - | |}
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| - |
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| - |
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| - | * Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
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| - | * Cut band out of gel (use UV box)
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| - |
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| - |
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| - | Results from gel recovery:
| |
| - | {| class="wikitable" border=1 cellpadding="5" cellspacing="0"
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| - | |-
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| - | | Sample|| 260|| 260/280 || ng/μL
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| - | |-
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| - | | H2B - X/S 1 || 0.016 ||1.807|| 15.961
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| - | |-
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| - | | H2B - X/S 2 || 0.009 || 1.976 || 8.619
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| - | |-
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| - | | LOV - X/S 1 || 0.006|| 1.694 || 6.455
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| - | |-
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| - | | LOV - X/S 2 || 0.025|| 1.883 || 25.363
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| - | |}
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| - |
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| - |
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| - | [[Image:VN_Gel_11-21-12.png|Results of from the digests are shown.]]
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| - | [[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
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| - |
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| - | # H2B = 410
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| - | # LOV = 461
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| - |
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| - | * There were two samples made of each PCR reaction. The two from the H2B are shown on the left, and the two from the LOV are shown on the right.
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| - |
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| - |
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| - | '''Dephosphorylation''' (11/23/12)
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| - |
| |
| - | > Dephosphorylation (Roche)
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| - | # pSB1A3 - X/S = 2000 bp
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| - |
| |
| - | {| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
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| - | |-
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| - | | <u>Reagent</u> || <u>Volume</u>
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| - | |-
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| - | | DNA (clean digest) || 11.0 (~200 ng)
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| - | |-
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| - | | 10x phos. buffer || 1.5
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| - | |-
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| - | | phosphatase || 0.5
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| - | |-
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| - | | dH<sub>2</sub>O || 2.0
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| - | |-
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| - | | || 15 μL
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| - | |}
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| - |
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| - | * Incubate at 37°C/ 10 min.
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| - | * Heat inactivate at 75°C/ 2 min.
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| - | * [final] = 200 ng/μL / 15 μL = ~13 ng/μL
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| - |
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| - |
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| - | '''Ligation'''
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| - | * Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3'''
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| - | * Use 3x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 16 ng/μL H2B = at least '''0.8 μL H2B'''
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| - | * Use 3x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 25 ng/μL LOV = at least '''0.5 μL LOV'''
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| - |
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| - |
| |
| - | {| class="wikitable" width=700px
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| - | | || H2B || LOV || Negative Control || H2B || LOV || Negative Control
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| - | |-
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| - | | Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || ---
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| - | |-
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| - | | Vector DNA (50 ng) || 1.5 || 1.5 || 1.5 || 1.5 || 1.5 || 1.5
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| - | |-
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| - | | 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0
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| - | |-
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| - | | T4 ligase || 1.0 NEB || 1.0 NEB || 1.0 NEB || 1.0 Roche || 1.0 Roche || 1.0 Roche
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| - | |-
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| - | | dH<sub>2</sub>O || 1.5 || 1.5 || 2.5 || 1.5 || 1.5 || 2.5
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| - | |-
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| - | | TOTAL || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0
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| - | |-
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| - | | colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.
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| - | |}
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| - |
| |
| - | Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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| - |
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