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Line 30: |
Line 30: |
| | bgcolor=#cfcfcf | Volume | | | bgcolor=#cfcfcf | Volume |
| | bgcolor=#cfcfcf | 5x mix | | | bgcolor=#cfcfcf | 5x mix |
| | | rowspan="7" | [[Image:KAH010613_gel1.jpg|270px|PCR gel]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] |
| |- | | |- |
| | Colony || --- || --- | | | Colony || --- || --- |
Line 49: |
Line 50: |
| * 72°C, 3 min. | | * 72°C, 3 min. |
| * 4°C, ∞ | | * 4°C, ∞ |
| | |
| | Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C) |
|
| |
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| |
|
| ---- | | ---- |
| '''Assemblies''' | | '''Transformation''' |
| # BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size | | # hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson |
| # BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
| | # V0120, 6.0 μL 1/4x Gibson |
| | | # hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson |
| | | # pSB1A3, 6.0 μL 1/4x Gibson |
| * 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.
| |
| |}
| |
| | |
| | |
| * Measure conc.'s
| |
| {| {{table}} cellspacing="3" <!-- [DNA] table -->
| |
| |- bgcolor=#cfcfcf
| |
| | Sample || OD260 || 260/280 || ng/μL
| |
| |-
| |
| | 1. Digested part (a/b) || --- || --- || ---
| |
| |-
| |
| | 2. Digested part (c/d) || --- || --- || ---
| |
| |}
| |
|
| |
|
|
| |
|
| * 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
| |
| {| {{table}} cellspacing="3" <!-- Ligations table -->
| |
| |- bgcolor=#cfcfcf
| |
| | Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
| |
| |-
| |
| | 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
| |
| |-
| |
| | 2. vector(c/d)/ ## ng ||
| |
| |}
| |
|
| |
| {| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| |
| | || 1 || 2 ||
| |
| |-
| |
| | Insert DNA || ### || --- ||
| |
| |-
| |
| | Vector DNA || ### || ### ||
| |
| |-
| |
| | 2x lgn buf (Roche) || ### || ### ||
| |
| |-
| |
| | T4 ligase (NEB) || 1.0 || 1.0 ||
| |
| |-
| |
| | dH<sub>2</sub>O || ### || ### ||
| |
| |-
| |
| | || # μL || # μL ||
| |
| |}
| |
|
| |
| ----
| |
| '''Oligo annealing'''
| |
| # New BB 1
| |
| # New BB 2
| |
|
| |
| {| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
| |
| | DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
| |
| |-
| |
| | 10x annealing buffer || 2.0
| |
| |-
| |
| | dH<sub>2</sub>O || ---
| |
| |-
| |
| | || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
| |
| |}
| |
|
| |
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| |
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