User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/06

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(01/06/13)
Current revision (19:38, 6 January 2013) (view source)
(01/06/13)
 
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| 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 || --- || ---
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* 4°C, ∞
* 4°C, ∞
 +
Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)
-
----
 
-
'''Assemblies'''
 
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
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* Digests (Fermentas FD)
 
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** Specific notes
 
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{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
 
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|- valign="top"
 
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| bgcolor=#cfcfcf | Reagent
 
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| bgcolor=#cfcfcf | Volume
 
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| 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] -->
 
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|-
 
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| DNA (plasmid) || up to 25 μL
 
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|-
 
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| 10x buffer || 3.0
 
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|-
 
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| enzyme 1 || 1.0
 
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|-
 
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| enzyme 2 || 1.0
 
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|-
 
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| dH<sub>2</sub>O || ---
 
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|-
 
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
 
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|}
 
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* Measure conc.'s
 
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{| {{table}} cellspacing="3" <!-- [DNA] table -->
 
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|- bgcolor=#cfcfcf
 
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| Sample || OD260 || 260/280 || ng/μL
 
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|-
 
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| 1. Digested part (a/b) || --- || --- || ---
 
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|-
 
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| 2. Digested part (c/d) || --- || --- || ---
 
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|}
 
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* Dephosphorylation (Roche)
 
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{| {{table}} cellspacing="3" <!-- Dephos table -->
 
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|-
 
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| bgcolor=#cfcfcf | Reagent
 
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| bgcolor=#cfcfcf | Volume
 
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|-
 
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| DNA (clean digest) || up to 17 μL (500 ng)
 
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|-
 
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| 10x buffer d.p. || 2.0
 
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|-
 
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| phosphatase || 1.0
 
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|-
 
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| dH<sub>2</sub>O || ---
 
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|-
 
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| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
 
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|}
 
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* Ligations
 
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{| {{table}} cellspacing="3" <!-- Ligations table -->
 
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|- bgcolor=#cfcfcf
 
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
 
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|-
 
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| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
 
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|-
 
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| 2. vector(c/d)/ ## ng || &nbsp;
 
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|}
 
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
 
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| &nbsp;            || 1    || 2    ||
 
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|-
 
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| Insert DNA        || ###  || ---  ||
 
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|-
 
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| Vector DNA        || ###  || ###  ||
 
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|-
 
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| 2x lgn buf (Roche) || ###  || ###  ||
 
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|-
 
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| T4 ligase (NEB)    || 1.0  || 1.0  ||
 
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|-
 
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| dH<sub>2</sub>O    || ###  || ###  ||
 
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|-
 
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| &nbsp;            || # μL || # μL ||
 
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|}
 
----
----
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'''Oligo annealing'''
+
'''Transformation'''
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# New BB 1
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# hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson
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# New BB 2
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# V0120, 6.0 μL 1/4x Gibson
 +
# hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson
 +
# pSB1A3, 6.0 μL 1/4x Gibson
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{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
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* Add DNA to 50 μL DH5α-Turbo; ice/ 10 min.
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| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
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* Plate on 100 μg/mL amp.
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|-
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| 10x annealing buffer || 2.0
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-
|-
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| dH<sub>2</sub>O || ---
+
-
|-
+
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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
+
-
|}
+

Current revision

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

01/06/13

  • Gibson Assembly: pick colonies & check via colony PCR
  • Media: Make LB agar +amp
  • Gibson Assembly: redo transformation with more DNA



Gibson Assembly results

  • hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
  • hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
  • hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
  • hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
  • Pick and streak colonies from plates 3, 5, 7
  • Use same pipette tip for colony PCR
Reagent Volume 5x mix PCR gel
30 μL/lane, 1% agarose; Ladder
Colony --- ---
10 μM primer BL9 1.0 5.0
10 μM primer BL10 1.0 5.0
2x GoTaq 12.5 62.5
dH2O 10.5 52.5
  25.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞

Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)



Transformation

  1. hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson
  2. V0120, 6.0 μL 1/4x Gibson
  3. hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson
  4. pSB1A3, 6.0 μL 1/4x Gibson
  • Add DNA to 50 μL DH5α-Turbo; ice/ 10 min.
  • Plate on 100 μg/mL amp.



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