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Line 6: |
Line 6: |
| | colspan="2"| | | | colspan="2"| |
| <!-- ##### 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. ##### --> |
| ==mm/dd/yy== | | ==08/26/15== |
| <!-- Precede finished items with a checkmark ✓ --> | | <!-- Precede finished items with a checkmark ✓ --> |
| * Line item 1 | | * Minipreps - CRISPR library |
| * Line item 2 | | * Cultures - CRISPR library (round 2) |
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|
| ---- | | ---- |
| '''Line item''' | | '''Minipreps - CRISPR library''' |
| | |
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| * Minipreps | | * Minipreps |
| ** Sigma GenElute kit, elute w/ 75 μL elution sln. | | ** Sigma GenElute kit, elute w/ 75 μL elution sln. |
| | | # L34-032 |
| * Digests
| | # L34-040 |
| ** Check with #/# digests
| | # L34-094 |
| | | # L34-096 |
| {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
| |
| |- valign="top"
| |
| | bgcolor=#cfcfcf | Reagent
| |
| | bgcolor=#cfcfcf | Volume
| |
| | rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| |
| | rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| |
| |-
| |
| | DNA(plasmid) || 2.0 μL
| |
| |-
| |
| | 10X buffer || 1.5
| |
| |-
| |
| | EcoRI || 1.0
| |
| |-
| |
| | PstI || 1.0
| |
| |-
| |
| | dH<sub>2</sub>O || 9.5
| |
| |-
| |
| | || 15 μL --> 37°C/ ~15 min.
| |
| |}
| |
| | |
| ----
| |
| '''Line item'''
| |
| | |
| | |
| * Assemblies
| |
| # BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
| |
| # BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size | |
| | |
| | |
| * Digests (Fermentas FD)
| |
| ** Specific notes
| |
| | |
| {| {{table}} cellspacing="3" <!-- Digest rxn. table -->
| |
| |- valign="top"
| |
| | bgcolor=#cfcfcf | Reagent
| |
| | bgcolor=#cfcfcf | Volume
| |
| | rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| |
| |-
| |
| | DNA (plasmid) || up to 25 μL
| |
| |-
| |
| | 10x buffer || 3.0
| |
| |-
| |
| | enzyme 1 || 1.0
| |
| |-
| |
| | enzyme 2 || 1.0
| |
| |-
| |
| | dH<sub>2</sub>O || ---
| |
| |-
| |
| | || 30 μL --> 37°C/ ~30 min.
| |
| |}
| |
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Line 83: |
Line 34: |
| |} | | |} |
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|
| | | * Sequencing |
| * Dephosphorylation (Roche) | |
| {| {{table}} cellspacing="3" <!-- Dephos table -->
| |
| |-
| |
| | bgcolor=#cfcfcf | Reagent
| |
| | bgcolor=#cfcfcf | Volume
| |
| |-
| |
| | DNA (clean digest) || up to 17 μL (500 ng)
| |
| |-
| |
| | 10x buffer d.p. || 2.0
| |
| |-
| |
| | phosphatase || 1.0
| |
| |-
| |
| | dH<sub>2</sub>O || ---
| |
| |-
| |
| | || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| |
| |}
| |
| | |
| | |
| * Ligations
| |
| # insert/(a/b)/size + vector/(c/d)/size
| |
| # vector/(c/d)/size
| |
| | |
| {| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| |
| |- valign="top"
| |
| | bgcolor=#cfcfcf | Reagent
| |
| | bgcolor=#cfcfcf | Rxn1
| |
| | bgcolor=#cfcfcf | Rxn2
| |
| |-
| |
| | Insert DNA || ### || ---
| |
| |-
| |
| | Vector DNA || ### || ###
| |
| |-
| |
| | 2x lgn buf (Roche) || ### || ###
| |
| |-
| |
| | T4 ligase (NEB) || 1.0 || 1.0
| |
| |-
| |
| | dH<sub>2</sub>O || ### || ###
| |
| |-
| |
| | || # μL || # μL
| |
| |}
| |
| | |
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