Haynes Lab:Notebook/Engineering PC-TFs/2013/01/25

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(01/25/13)
(01/25/13)
(6 intermediate revisions not shown.)
Line 18: Line 18:
# pSB1A3, gg0001, gg0002
# pSB1A3, gg0001, gg0002
-
# template, primer 1, primer 2
+
# hPCD, gg0003, gg0004
-
# template, primer 1, primer 2
+
# BL02, gg0005, gg0007
-
# template, primer 1, primer 2
+
# BL03, gg0005, gg0008
 +
# BL04, gg0005, gg0009
 +
# fshPCD, gg0010, gg0012
Line 27: Line 29:
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
-
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
+
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL02 = 1431<br> 4. BL03 = 1731<br> 5. BL04 = 1686<br> 6.fshPCD = 186<br>
-
| rowspan="7" | [[Image:image.jpg|200px|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
+
| rowspan="7" | [[Image:gg_gel1-25-2013.jpg|200px|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 0.2 μL
| DNA(plasmid) || 0.2 μL
Line 49: Line 51:
-
Purification
+
Purification (Completed on 1-25-2013)
-
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
+
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O  
-
* Measure [DNA]
+
 
 +
* Measure [DNA] (1-29-2013)
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
|- valign="top"
Line 59: Line 62:
| bgcolor=#cfcfcf | ng/μL  
| bgcolor=#cfcfcf | ng/μL  
|-
|-
-
| 1. pSB1A3 || ### || ### || ###
+
| 1. pSB1A3 || .084|| 1.801 || 83.954
 +
|-
 +
| 2. hPCD || .017 || 1.734|| 17.344
 +
|-
 +
| 3. BL02 || .058 || 1.764|| 58.413
 +
|-
 +
| 4. BL03 || .082 || 1.831|| 81.877
|-
|-
-
| 2. ### || ### || ### || ###
+
| 5. BL04 || .05 || 1.888 || 49.561
|-
|-
-
| 3. ### || ### || ### || ###
+
| 6. fshPCD || .021 || 1.622 || 21.378
|}
|}

Revision as of 02:52, 1 February 2013

Engineering PC-TFs Main project page
Previous entry      Next entry

01/25/13

  • Type IIS assembly - day 1



PCR

  • Resuspend new primers to a final concentration of 100 μM
  • Make 10 μM dilutions in separate tubes
  • All of the reactions are as follows:
  1. pSB1A3, gg0001, gg0002
  2. hPCD, gg0003, gg0004
  3. BL02, gg0005, gg0007
  4. BL03, gg0005, gg0008
  5. BL04, gg0005, gg0009
  6. fshPCD, gg0010, gg0012


Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL02 = 1431
4. BL03 = 1731
5. BL04 = 1686
6.fshPCD = 186
PCR results
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL
  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


Purification (Completed on 1-25-2013)

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA] (1-29-2013)
Sample OD 260 260/280 ng/μL
1. pSB1A3 .084 1.801 83.954
2. hPCD .017 1.734 17.344
3. BL02 .058 1.764 58.413
4. BL03 .082 1.831 81.877
5. BL04 .05 1.888 49.561
6. fshPCD .021 1.622 21.378



Personal tools