User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/01: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(3 intermediate revisions by the same user not shown)
Line 21: Line 21:
# KAH87
# KAH87
# MV10
# MV10
* Measure conc.'s of minipreps
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. KAH87 (X/S) || 0.180 || 1.923 || 179.8
|-
| 2. MV10 (X) || 0.371 || 1.926 || 370.5
|}




Line 36: Line 47:
| bgcolor=#cfcfcf | Rxn 1
| bgcolor=#cfcfcf | Rxn 1
| bgcolor=#cfcfcf | Rxn 2
| bgcolor=#cfcfcf | Rxn 2
| 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:KAH050115_gel1.jpg|200px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (plasmid) || 20.0 || 15.0
| DNA (plasmid) || 20.0 || 15.0
Line 48: Line 59:
| dH<sub>2</sub>O || 5.0 || 10.0
| dH<sub>2</sub>O || 5.0 || 10.0
|-
|-
| &nbsp; || 30 μL  
| &nbsp; || 30 μL ||
|}
|}
--> 37°C/ ~30 min.
--> 37°C/ ~30 min.
Line 63: Line 74:
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. KAH87 (X/S) || --- || --- || ---
| 1. KAH87 (X/S) || 0.003 || 1.571 || 3.49
|-
|-
| 2. MV10 (X) || --- || --- || ---
| 2. MV10 (X) || 0.070 || 1.843 || 70.41
|}
|}


Line 77: Line 88:
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| DNA (clean digest) || 7.1 (500 ng)
|-
|-
| 10x buffer d.p. || 2.0
| 10x buffer d.p. || 2.0
Line 83: Line 94:
| phosphatase || 1.0
| phosphatase || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 9.9
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| &nbsp; || 20 μL  
|}
|}
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL




* Ligations
* Ligations
** 2:1 ratio calc.: 1053 (KAH87) / 5197 (MV10) * 2 * 50 = '''20 ng KAH87'''
# KAH87 (X/S) + MV10 (X)
# KAH87 (X/S) + MV10 (X)
# MV10 (X dp)
# MV10 (X dp), neg ctrl.


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
| Insert DNA        || ### || ---  ||
| Insert DNA        || 5.7 || ---  ||
|-
|-
| Vector DNA        || ### || ### ||
| Vector DNA        || 2.0 || 2.0 ||
|-
|-
| 2x lgn buf (Roche) || ### || ### ||
| 2x lgn buf (Roche) || 8.7 || 8.7 ||
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
|-
| dH<sub>2</sub>O    || ### || ### ||
| dH<sub>2</sub>O    || --- || 5.7 ||
|-
|-
| &nbsp;            || # μL || # μL ||
| &nbsp;            || 17.4 μL || 17.4 μL ||
|}
|}


Revision as of 13:37, 4 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

05/01/15

  • Assembly: KAH87 (hPcTF ORF) into MV10


Assembly: KAH87 (hPcTF ORF) into MV10


  • Minipreps (5 mL cultures)
    • Sigma GenElute kit
    • Elute w/ 75 μL elution sln.
  1. KAH87
  2. MV10


  • Measure conc.'s of minipreps
Sample OD260 260/280 ng/μL
1. KAH87 (X/S) 0.180 1.923 179.8
2. MV10 (X) 0.371 1.926 370.5


  • Assemblies
  1. KAH87/MV10: KAH87/(X/S)/1053 + MV10/(X)/5197


  • Digests (Fermentas FD)
  1. KAH87, X/S
  2. MV10, X
Reagent Rxn 1 Rxn 2 Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 15.0
10x buffer 3.0 3.0
XbaI 1.0 1.0
SpeI 1.0 ---
dH2O 5.0 10.0
  30 μL

--> 37°C/ ~30 min.


  • Gel purify
    • Sigma kit
    • Elute & back-elute w/ 25 μL elution sln.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. KAH87 (X/S) 0.003 1.571 3.49
2. MV10 (X) 0.070 1.843 70.41


  • Dephosphorylation (Roche)
    • Dephos the MV10 (X) fragment
Reagent Volume
DNA (clean digest) 7.1 (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O 9.9
  20 μL

--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
    • 2:1 ratio calc.: 1053 (KAH87) / 5197 (MV10) * 2 * 50 = 20 ng KAH87
  1. KAH87 (X/S) + MV10 (X)
  2. MV10 (X dp), neg ctrl.
  1 2
Insert DNA 5.7 ---
Vector DNA 2.0 2.0
2x lgn buf (Roche) 8.7 8.7
T4 ligase (NEB) 1.0 1.0
dH2O --- 5.7
  17.4 μL 17.4 μL



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/05/01&oldid=883187"