User:Karmella Haynes/Notebook/BioBrick cloning/2012/12/11: Difference between revisions

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==12/11/12==
==12/11/12==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Note: colonies from retransformation of pSB1A3-RFP look red
* Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids)
* Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids)
* Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning
* Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning




----
'''Minipreps'''<br>
* pSB1A3-RFP (retransformation from 12/10/12)
* Concentrations: (1) 260 = , 260/280 = , '''ng/μL = '''; (2) 260 = , 260/280 = , '''ng/μL = ''';
* Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>3. pSB1A3 = size<br>4. BB2 = size<br>
| rowspan="7" | [[Image:KAH121112_gel1.png|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA(plasmid) || 10.0 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'''
'''Assemblies'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# KAH111/C-His: 5' part/(E/P)/1123 + C-His/(E/P)/3600
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size




* Digests (Fermentas FD)
* Digests (Fermentas FD)
** Specific notes


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
Line 51: Line 25:
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| 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] -->
| rowspan="7" | [[Image:KAH121112_gel2.png|200px|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
| DNA (plasmid) || 15.0 μL
|-
|-
| 10x buffer || 3.0
| 10x buffer || 3.0
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| enzyme 2 || 1.0
| enzyme 2 || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 10.0
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 30 μL --> 37°C/ ~30 min.
Line 67: Line 41:




* Measure conc.'s
* Measure conc.'s of DNA purified from gel (Zymo kit)
{| {{table}} cellspacing="3" <!-- [DNA] table -->
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf  
|- bgcolor=#cfcfcf  
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. KAH11 (E/P) || 0.019 || 1.96 || 18.7
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 2. C-His (E/P) || 0.047 || 1.82 || 46.8
|}
 
 
* 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 || ---
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|}
|}


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{| {{table}} cellspacing="3" <!-- Ligations table -->
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
|- bgcolor=#cfcfcf
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 12/12/12</font>
|-
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
| 1. KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 25ng || <font color="blue">KAH111/C-His 3:1 (Pick 4)</font> (very small colonies after ~18 hours)
|-
|-
| 2. vector(c/d)/ ## ng || &nbsp;
| 2. C-His(E/P)/ 25 ng || 3 colonies
|}
|}


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2   ||
| &nbsp;            || 1    || 2  
|-
|-
| Insert DNA        || ### || ---  ||
| Insert DNA        || 1.0 || ---   
|-
|-
| Vector DNA        || ### || ### ||
| Vector DNA        || 0.5 || 0.5  
|-
|-
| 2x lgn buf (Roche) || ###  || ### ||
| 2x lgn buf (Roche) || 5.0 || 5.0
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0   
|-
|-
| dH<sub>2</sub>O    || ### || ### ||
| dH<sub>2</sub>O    || 2.5 || 3.5  
|-
|-
| &nbsp;            || # μL || # μL ||
| &nbsp;            || 10 μL || 10 μL  
|}
|}


----
----
'''Oligo annealing'''
'''Minipreps'''<br>
# New BB 1
* pSB1A3-RFP (retransformation from 12/10/12)
# New BB 2
* Note: pellets are red
* Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)
* Note: combined mini preps afterwards; concentration = 60 ng/μL


{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>3. pSB1A3 = 2155<br>4. pSB1A3 = 2155<br>
| rowspan="7" | [[Image:KAH121112_gel1.png|200px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA(plasmid) || 10.0 μL
|-
|-
| 10x annealing buffer || 2.0
| 10X buffer || 3.0
|-
|-
| dH<sub>2</sub>O || ---
| XbaI || 1.0
|-
|-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
| SpeI || 1.0
|-
| dH<sub>2</sub>O || 15.0
|-
| &nbsp; || 15 μL --> 37°C/ ~30 min.
|}
|}
* Gel purification: combined the two slices
** Concentration: 260 = 0.015, 260/280 = 2.2, '''ng/μL = 15.1'''




Revision as of 11:27, 12 December 2012

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12/11/12

  • Note: colonies from retransformation of pSB1A3-RFP look red
  • Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids)
  • Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning


Assemblies

  1. KAH111/C-His: 5' part/(E/P)/1123 + C-His/(E/P)/3600


  • Digests (Fermentas FD)
Reagent Volume Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 15.0 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 10.0
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s of DNA purified from gel (Zymo kit)
Sample OD260 260/280 ng/μL
1. KAH11 (E/P) 0.019 1.96 18.7
2. C-His (E/P) 0.047 1.82 46.8


  • Ligations
Ligation Plate results (lig : neg crtl) 12/12/12
1. KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 25ng KAH111/C-His 3:1 (Pick 4) (very small colonies after ~18 hours)
2. C-His(E/P)/ 25 ng 3 colonies
  1 2
Insert DNA 1.0 ---
Vector DNA 0.5 0.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.5 3.5
  10 μL 10 μL


Minipreps

  • pSB1A3-RFP (retransformation from 12/10/12)
  • Note: pellets are red
  • Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)
  • Note: combined mini preps afterwards; concentration = 60 ng/μL
Reagent Volume Expected:
3. pSB1A3 = 2155
4. pSB1A3 = 2155
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 10.0 μL
10X buffer 3.0
XbaI 1.0
SpeI 1.0
dH2O 15.0
  15 μL --> 37°C/ ~30 min.


  • Gel purification: combined the two slices
    • Concentration: 260 = 0.015, 260/280 = 2.2, ng/μL = 15.1



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