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

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(Autocreate 2013/01/06 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
Current revision (20:38, 6 January 2013) (view source)
(01/06/13)
 
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| colspan="2"|
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==mm/dd/yy==
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==01/06/13==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
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* Gibson Assembly: pick colonies & check via colony PCR
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* Line item 2
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* Media: Make LB agar +amp
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* Gibson Assembly: redo transformation with more DNA
----
----
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'''Minipreps'''<br>
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'''Gibson Assembly results'''<br>
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* Check with E/P digests
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* Plates from [http://openwetware.org/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/05 01/05/2013]
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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* hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
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|- valign="top"
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* hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
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| bgcolor=#cfcfcf | Reagent
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* hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
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| bgcolor=#cfcfcf | Volume
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* hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
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| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
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| 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] -->
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|-
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| DNA(plasmid) || 2.0 μL
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|-
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| 10X buffer || 1.5
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|-
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| EcoRI || 1.0
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|-
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| PstI || 1.0
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|-
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| dH<sub>2</sub>O || 9.5
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|-
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
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|}
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----
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'''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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* Pick and streak colonies from plates 3, 5, 7
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* Use same pipette tip for colony PCR
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* Dephosphorylation (Roche)
 
{| {{table}} cellspacing="3" <!-- Dephos table -->
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
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| bgcolor=#cfcfcf | 5x mix
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| 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]
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|-
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| DNA (clean digest) || up to 17 μL (500 ng)
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| Colony || --- || ---
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|-
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| 10x buffer d.p. || 2.0
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| 10 μM primer BL9 || 1.0 || 5.0
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|-
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| phosphatase || 1.0
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| 10 μM primer BL10 || 1.0 || 5.0
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|-
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| dH<sub>2</sub>O || ---
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| 2x GoTaq || 12.5 || 62.5
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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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| dH<sub>2</sub>O || 10.5 || 52.5
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|-
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| &nbsp; || 25.0 μL ||
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|}
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PCR
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* 95°C, 3 min.
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* [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
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* 72°C, 3 min.
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* 4°C, ∞
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* Ligations
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Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)
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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'''
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'''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
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# hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson
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# 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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|-
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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
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|}
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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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