Team:Lethbridge/Notebook/Lab Work/August
From 2010.igem.org
(→Aug 14/2010 Evening) |
(→August) |
||
Line 422: | Line 422: | ||
</table><br> | </table><br> | ||
- | ==<font color="white">August 6 | + | ==<font color="white">August 6, 2010== |
In Lab: JV | In Lab: JV | ||
Line 460: | Line 460: | ||
Gel extraction was carried out using QIAGEN method. Eluted to 12 (µL). | Gel extraction was carried out using QIAGEN method. Eluted to 12 (µL). | ||
- | ==<font color="white">August 6 | + | ==<font color="white">August 6, 2010 Evening== |
<b>Objective:</b> Attempt colony pcr for rapid screening<br> | <b>Objective:</b> Attempt colony pcr for rapid screening<br> | ||
Line 544: | Line 544: | ||
*ran at 100V for 75 minutes | *ran at 100V for 75 minutes | ||
- | ==<font color="white">Aug 9 | + | ==<font color="white">Aug 9, 2010== |
(In Lab: HB) | (In Lab: HB) | ||
Line 688: | Line 688: | ||
---- | ---- | ||
- | ==<font color="white">Aug 9 | + | ==<font color="white">Aug 9,2010 Evening== |
(In Lab: JV, AS)<br> | (In Lab: JV, AS)<br> | ||
Line 719: | Line 719: | ||
Incubated reactions overnight at room temperature. | Incubated reactions overnight at room temperature. | ||
- | ==<font color="white">Aug 10 | + | ==<font color="white">Aug 10, 2010== |
(In Lab: JV)<br> | (In Lab: JV)<br> | ||
Line 778: | Line 778: | ||
- | ==<font color="white">Aug 13 | + | ==<font color="white">Aug 13, 2010== |
(In Lab: AS)<br> | (In Lab: AS)<br> | ||
Line 799: | Line 799: | ||
Added 49µL Master Mix to each reaction tube. | Added 49µL Master Mix to each reaction tube. | ||
- | ==<font color="white">Aug 14 | + | ==<font color="white">Aug 14, 2010== |
(In Lab: AS)<br> | (In Lab: AS)<br> | ||
Line 868: | Line 868: | ||
GEL PICTURE! | GEL PICTURE! | ||
- | ==<font color="white">Aug 14 | + | ==<font color="white">Aug 14, 2010 Evening== |
(In Lab: AS)<br> | (In Lab: AS)<br> | ||
Line 948: | Line 948: | ||
Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 20 minutes at 80<sup>o</sup>C. | Incubated at 37<sup>o</sup>C for 1.5 hours. Heat killed enzymes for 20 minutes at 80<sup>o</sup>C. | ||
+ | |||
+ | <u>Ligation Reactions:</u> | ||
+ | |||
+ | 3 Part: Lumazine/mms6 + dT + psB1C3 | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b><td><b>1X(µL)</b><td><b>Master Mix(x5.5)(µL)</b> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td>11.0<td>64.9 | ||
+ | <tr><td>T4 Ligase Buffer (10x)</td><td>2<td>11 | ||
+ | <tr><td>Plasmid (psB1C3)</td><td>2<td>11 | ||
+ | <tr><td>Part 1 (Lumazine/mms6)</td><td>2 | ||
+ | <tr><td>Part 2 (dT)</td><td>2<td>11 | ||
+ | <tr><td>T4 DNA Ligase</td><td>0.2<td>1.1 | ||
+ | </table> | ||
+ | |||
+ | 2 Part: Lumazine/mms6 + dT | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b><td><b>1X(µL)</b><td><b>Master Mix(x5.5)(µL)</b> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td>13.8<td>75.9 | ||
+ | <tr><td>T4 Ligase Buffer (10x)</td><td>2<td>11 | ||
+ | <tr><td>Part 1 (Lumazine/mms6)</td><td>2 | ||
+ | <tr><td>Part 2 (dT)</td><td>2<td>11 | ||
+ | <tr><td>T4 DNA Ligase</td><td>0.2<td>1.1 | ||
+ | </table> | ||
+ | |||
+ | Added 18(µL) to each rxn tube. Incubated 1 hour and overnight at room temperature ( 25<sup>o</sup>C). | ||
+ | |||
+ | |||
+ | <u>Screening via PCR amplification :</u> Thermocycler set to iGEM program 11<br> | ||
+ | 3 Part: Lum/mms6 + dT + pSB1C3 | ||
+ | <table border ="3"> | ||
+ | <tr><td><b>Component</b><td><b>1X(µL)</b><td><b>Master Mix(x5.5)(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>33.8<td>185.9 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>27.5 | ||
+ | <tr><td>dNTPs<td>2<td>11 | ||
+ | <tr><td>VF2 Primer<td>2<td>11 | ||
+ | <tr><td>VR Primer<td>2<td>11 | ||
+ | <tr><td>Template DNA<td>5<td> | ||
+ | <tr><td>Pfu polymerase<td>0.2<td>1.1 | ||
+ | </table><br> | ||
+ | |||
+ | Added 45 (µL) MM to each tube. | ||
+ | |||
+ | 2 Part: Lum/mms6 + dT | ||
+ | <table border ="3"> | ||
+ | <tr><td><b>Component</b><td><b>1X(µL)</b><td><b>Master Mix(x5.5)(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>6.8<td>37.4 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>11 | ||
+ | <tr><td>dNTPs<td>2<td>11 | ||
+ | <tr><td>Prefix Primer<td>2<td>11 | ||
+ | <tr><td>Suffix Antisense Primer<td>2<td>11 | ||
+ | <tr><td>Template DNA<td>5<td> | ||
+ | <tr><td>Pfu polymerase<td>0.2<td>1.1 | ||
+ | </table><br> | ||
+ | |||
+ | Added 15 (µL) MM to each tube. |