IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/08/04

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Current revision (22:33, 6 August 2014) (view source)
(Preparing another RBS-TesA and LacI Ligation)
 
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==Preparing another RBS-TesA and LacI Ligation==
==Preparing another RBS-TesA and LacI Ligation==
* Digested more of RBS-TesA with X and P(insert). Digested more of LacI with S and P (backbone).
* Digested more of RBS-TesA with X and P(insert). Digested more of LacI with S and P (backbone).
 +
: TesA+RBS insert with LacI+backbone
 +
{| {{table}} border="1" align=center <!-- Digestion table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | RBS-TesA (μL)
 +
| bgcolor=#cfcfcf | LacI (μL)
 +
|-
 +
|| DNA || 15.0|| 15.0
 +
|-
 +
|| 10X fast Digest Buffer|| 2.0  || 2.0
 +
|-
 +
|| PstI || 1.0  || 1.0
 +
|-
 +
|| XbaI|| 1.0 || .0
 +
|-
 +
|| SpeI || 0.0 || 1.0
 +
|-
 +
|| H<sub>2</sub>O || 1.0 || 1.0
 +
|-
 +
|| 37°C for 1 hour
 +
|-
 +
 +
|}
 +
 +
*Both samples used in the Clean and Concentrate kit
*Treat the LacI with Antarctic Phsophotase.  
*Treat the LacI with Antarctic Phsophotase.  
 +
: Antarctic Phsophotase Treatment
 +
{| {{table}} border="1" align=center <!-- Digestion table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf |
 +
|-
 +
|| LacI DNA || 10.0
 +
|-
 +
|| Antarctic Phosphotase|| 1.0 
 +
|-
 +
|| Phosphotase Buffer || 2.0 
 +
|-
 +
|| H<sub>2</sub>O || 7.0
 +
|-
 +
|| 37°C for 1 hour then 65°C for 5 min
 +
|-
 +
 +
|}
*The RBS-TesA and LacI DNA samples were quantified:
*The RBS-TesA and LacI DNA samples were quantified:
-
RBS-TesA: 39.157 ng/μL
+
*RBS-TesA: 39.157 ng/μL
-
LacI: 62.579 ng/μL
+
*LacI: 62.579 ng/μL
*Overnight ligation reactions were conducted for the test ligation and the negative control.
*Overnight ligation reactions were conducted for the test ligation and the negative control.
 +
: TesA+RBS insert with LacI+backbone
 +
{| {{table}} border="1" align=center <!-- Digestion table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | 1 (μL)
 +
| bgcolor=#cfcfcf | Control (μL)
 +
|-
 +
|| TesA+RBS || 1.25 || 0.0
 +
|-
 +
|| LacI || 1.0  || 1.0
 +
|-
 +
|| Ligation Buffer || 1.0  || 1.0
 +
|-
 +
|| Ligase || 1.0 || 1.0
 +
|-
 +
|| H<sub>2</sub>O || 5.75 || 7.0
 +
|-
 +
|| 16°C overnight
 +
|-
 +
 +
|}
 +
 +
<u>Ethanol:</u>
 +
 +
*Started seeds of 3 seperate colonies of ethanol plasmid
 +
**50 ml LB w/ Chloramphenicol + picked colony
 +
**Cells didn't grow fast enough to add IPTG to start protein growth, so cells were left overnight to grow more
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Preparing another RBS-TesA and LacI Ligation

  • Digested more of RBS-TesA with X and P(insert). Digested more of LacI with S and P (backbone).
TesA+RBS insert with LacI+backbone
Reagent RBS-TesA (μL) LacI (μL)
DNA 15.0 15.0
10X fast Digest Buffer 2.0 2.0
PstI 1.0 1.0
XbaI 1.0 .0
SpeI 0.0 1.0
H2O 1.0 1.0
37°C for 1 hour
  • Both samples used in the Clean and Concentrate kit
  • Treat the LacI with Antarctic Phsophotase.
Antarctic Phsophotase Treatment
Reagent
LacI DNA 10.0
Antarctic Phosphotase 1.0
Phosphotase Buffer 2.0
H2O 7.0
37°C for 1 hour then 65°C for 5 min
  • The RBS-TesA and LacI DNA samples were quantified:
  • RBS-TesA: 39.157 ng/μL
  • LacI: 62.579 ng/μL
  • Overnight ligation reactions were conducted for the test ligation and the negative control.
TesA+RBS insert with LacI+backbone
Reagent 1 (μL) Control (μL)
TesA+RBS 1.25 0.0
LacI 1.0 1.0
Ligation Buffer 1.0 1.0
Ligase 1.0 1.0
H2O 5.75 7.0
16°C overnight

Ethanol:

  • Started seeds of 3 seperate colonies of ethanol plasmid
    • 50 ml LB w/ Chloramphenicol + picked colony
    • Cells didn't grow fast enough to add IPTG to start protein growth, so cells were left overnight to grow more
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