Difference between revisions of "Team:Potsdam/notebook/lowcopy"
| Line 144: | Line 144: | ||
gel picture: <br> | gel picture: <br> | ||
<img src="2017.igem.org/wiki/images/c/c5/Gel14_8.tiff" width="50%"> | <img src="2017.igem.org/wiki/images/c/c5/Gel14_8.tiff" width="50%"> | ||
| − | + | <br> | |
- order of pockets: ABC, A and B are correct size, C did not work well, there is a band at the desired result and one at the unopened plasmid (and several other stuff) so PCR has to be done again <br> | - order of pockets: ABC, A and B are correct size, C did not work well, there is a band at the desired result and one at the unopened plasmid (and several other stuff) so PCR has to be done again <br> | ||
- dCas9 fragments look good and can be worked with tomorrow <br> | - dCas9 fragments look good and can be worked with tomorrow <br> | ||
<br> <br> | <br> <br> | ||
| + | <b> Tuesday, 08/15/17 </b> | ||
| + | <br> <br> | ||
| + | 1. DpnI digestion of dCas9 fragments A + B <br> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <th width="50%" align="center"><b>component</b></th> | ||
| + | <th width="50%" align="center"><b>volume in µl</b></th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">DpnI</td> | ||
| + | <td align="center">2,6</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">DNA</td> | ||
| + | <td align="center">6,5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">cutsmart-Buffer</td> | ||
| + | <td align="center">5,0</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">H<sub>2</sub>O</td> | ||
| + | <td align="center">35,9</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">final volume</td> | ||
| + | <td align="center">50</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | - Duration: 1 hour <br><br> | ||
| + | <b>IMPORTANT: Fabian told me you can just add about 1 uL DpnI to the reaction-mix </b> | ||
| + | <br><br> | ||
| + | 2. Gel run of dCas9 A + B <br> | ||
| + | <div style="text-align: justify; margin-left:20px"> | ||
| + | - let it run on gel for purification <br> | ||
| + | - 140 V a 40 min <br> | ||
| + | - cut out was done by Natalie and Pauline </div> | ||
| + | <br> | ||
| + | 3. Linearization PCR of psB4A5 (second atempt) via gradient PCR <br> | ||
| + | <div style="text-align: justify; margin-left:20px"> | ||
| + | - made new PCR using Q15/Q16 primers and psB4A5 plasmid fpr linearization <br> | ||
| + | - used 3 different DNA concentrations and 3 different annealing temperatures (9 reactions total)<br> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <th align="center"><b>component</b></th> | ||
| + | <th align="center"><b> 1 reaction: <br> volume in µl</b></th> | ||
| + | <th align="center"><b> 10 reaction MM: <br> volume in µl</b></th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">HiQ</td> | ||
| + | <td align="center">5</td> | ||
| + | <td align="center">50</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">primer</td> | ||
| + | <td align="center">1,25</td> | ||
| + | <td align="center">12,5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">DNA</td> | ||
| + | <td align="center">variable (1 µl)</td> | ||
| + | <td align="center">variable</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">H<sub>2</sub>O</td> | ||
| + | <td align="center">17,5</td> | ||
| + | <td align="center">175</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">final volume</td> | ||
| + | <td align="center">25</td> | ||
| + | <td align="center">250</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | - annealing temperatures: A = 60°C/B = 64,5°C/C = 68°C <br> | ||
| + | - DNA concentrations: 0,1 = 1:1000 / 1 = 1:100 / 10 = 1:10 <br> | ||
| + | Scheme:<br> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <th align="center"><b></b></th> | ||
| + | <th align="center"><b></b></th> | ||
| + | <th align="center"><b></b></th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">0,1 A</td> | ||
| + | <td align="center">0,1 B</td> | ||
| + | <td align="center">0,1 C</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">1 A</td> | ||
| + | <td align="center">1 B</td> | ||
| + | <td align="center">1 C</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">10 A</td> | ||
| + | <td align="center">10 B</td> | ||
| + | <td align="center">10 C</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <br> <br> | ||
</p> | </p> | ||
</div> | </div> | ||
Revision as of 10:58, 1 November 2017
Lab book
We splitted our labbook into the following parts:
Friday, 07/21/17
- took pSB4A5 backbone out of DNA distribution Kit and transformed into E.coli (rest from Kit was put in -20 °C)
Tuesday, 07/25/17
- Inoculated LB media with psB4A5
Wednesday, 07/26/17
- Miniprep with Promega “Plus SV Miniprep DNA Purification System”
- 2 approaches with psB4A5
- final concentration:
- final concentration:
- approach 1: 30 ng/ul
- approach 2: 40 ng/ul
- approach 2: 40 ng/ul
Thursday, 08/10/17
- glycerol stock (30%) of IG_C_P1_G (pSB4A5)
- prepared 10 mL preculture with pdCas9-Bacteria E.coli from plate from Fabian for Miniprep
Monday, 08/14/17
- primer resuspended to 100 uM (except Q8):
| primer | volume in µl |
|---|---|
| Q5 | 354 |
| Q6 | 957 |
| Q7 | 749 |
| Q8 | 657 (83,1 uM) |
| Q15 | 956 |
| Q16 | 1455 |
| Q17 | 1693 |
| Q18 | 1440 |
- prepareed 3 separate PCRs with primer pairs:
Q5/Q8 (Eppi A), Q6/Q7 (Eppi B), Q15/Q16 (Eppi c)
- pipetting scheme:
Q5 2X MasterMix: 5 uL
10 uM Primer: each 0,5 uL
Template: 1 uL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 uL
total: 10 uL
- cycling conditions (prog01 in Epepndorf Mastercycler nexus gradient (Torsten):10 uM Primer: each 0,5 uL
Template: 1 uL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 uL
total: 10 uL
| temperature | time in sec |
|---|---|
| 98 °C | 30 |
| 98 °C | 10 |
| 64 °C | 20 |
| 72 °C | 110 |
| 72 °C | 120 |
- 2.5 uL DNA + 2uL Loading Dye + 7.5 uL (total 12 uL)
- ran gel at 120 V for 40 minutes
gel picture:
- order of pockets: ABC, A and B are correct size, C did not work well, there is a band at the desired result and one at the unopened plasmid (and several other stuff) so PCR has to be done again
- dCas9 fragments look good and can be worked with tomorrow
Tuesday, 08/15/17
1. DpnI digestion of dCas9 fragments A + B
| component | volume in µl |
|---|---|
| DpnI | 2,6 |
| DNA | 6,5 |
| cutsmart-Buffer | 5,0 |
| H2O | 35,9 |
| final volume | 50 |
IMPORTANT: Fabian told me you can just add about 1 uL DpnI to the reaction-mix
2. Gel run of dCas9 A + B
- let it run on gel for purification
- 140 V a 40 min
- cut out was done by Natalie and Pauline
- 140 V a 40 min
- cut out was done by Natalie and Pauline
3. Linearization PCR of psB4A5 (second atempt) via gradient PCR
- made new PCR using Q15/Q16 primers and psB4A5 plasmid fpr linearization
- used 3 different DNA concentrations and 3 different annealing temperatures (9 reactions total)
- annealing temperatures: A = 60°C/B = 64,5°C/C = 68°C
- DNA concentrations: 0,1 = 1:1000 / 1 = 1:100 / 10 = 1:10
Scheme:
- used 3 different DNA concentrations and 3 different annealing temperatures (9 reactions total)
| component | 1 reaction: volume in µl |
10 reaction MM: volume in µl |
|---|---|---|
| HiQ | 5 | 50 |
| primer | 1,25 | 12,5 |
| DNA | variable (1 µl) | variable |
| H2O | 17,5 | 175 |
| final volume | 25 | 250 |
- DNA concentrations: 0,1 = 1:1000 / 1 = 1:100 / 10 = 1:10
Scheme:
| 0,1 A | 0,1 B | 0,1 C |
| 1 A | 1 B | 1 C |
| 10 A | 10 B | 10 C |
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