Difference between revisions of "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)
 
(01/06/13)
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| 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==
+
==01/06/13==
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
+
* Gibson Assembly: pick colonies & check via colony PCR
* Line item 2
+
* Media: Make LB agar +amp
 +
* Gibson Assembly: redo transformation with more DNA
  
  
 
----
 
----
'''Minipreps'''<br>
+
'''Gibson Assembly results'''<br>
* Check with E/P digests
+
* Plates from [http://openwetware.org/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/05 01/05/2013]
  
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
+
* hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
|- valign="top"
+
* hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
| bgcolor=#cfcfcf | Reagent
+
* hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
| bgcolor=#cfcfcf | Volume
+
* hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
| 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
 
|-
 
| &nbsp; || 15 μL --> 37°C/ ~15 min.
 
|}
 
 
 
----
 
'''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 || ---
 
|-
 
| &nbsp; || 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) || --- || --- || ---
 
|}
 
  
 +
* Pick and streak colonies from plates 3, 5, 7
 +
* Use same pipette tip for colony PCR
  
* Dephosphorylation (Roche)
 
 
{| {{table}} cellspacing="3" <!-- Dephos table -->
 
{| {{table}} cellspacing="3" <!-- Dephos table -->
 
|-
 
|-
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Volume
 
| bgcolor=#cfcfcf | Volume
 +
| 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]
 
|-
 
|-
| DNA (clean digest) || up to 17 μL (500 ng)
+
| Colony || --- || ---
 
|-
 
|-
| 10x buffer d.p. || 2.0
+
| 10 μM primer BL9 || 1.0 || 5.0
 
|-
 
|-
| phosphatase || 1.0
+
| 10 μM primer BL10 || 1.0 || 5.0
 
|-
 
|-
| dH<sub>2</sub>O || ---
+
| 2x GoTaq || 12.5 || 62.5
 
|-
 
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
+
| dH<sub>2</sub>O || 10.5 || 52.5
 +
|-
 +
| &nbsp; || 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, ∞
  
* Ligations
+
Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)
{| {{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 || &nbsp;
 
|}
 
  
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
 
| &nbsp;            || 1    || 2    ||
 
|-
 
| Insert DNA        || ###  || ---  ||
 
|-
 
| Vector DNA        || ###  || ###  ||
 
|-
 
| 2x lgn buf (Roche) || ###  || ###  ||
 
|-
 
| T4 ligase (NEB)    || 1.0  || 1.0  ||
 
|-
 
| dH<sub>2</sub>O    || ###  || ###  ||
 
|-
 
| &nbsp;            || # μL || # μL ||
 
|}
 
  
 
----
 
----
'''Oligo annealing'''
+
'''Transformation'''
# New BB 1
+
# hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson
# New BB 2
+
# V0120, 6.0 μL 1/4x Gibson
 +
# hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson
 +
# pSB1A3, 6.0 μL 1/4x Gibson
  
{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
+
* Add DNA to 50 μL DH5α-Turbo; ice/ 10 min.
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
+
* Plate on 100 μg/mL amp.
|-
 
| 10x annealing buffer || 2.0
 
|-
 
| dH<sub>2</sub>O || ---
 
|-
 
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
|}
 
  
  

Revision as of 16:38, 6 January 2013

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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.