Difference between revisions of "Team:TPHS San Diego/Notebook"
| Line 204: | Line 204: | ||
We want to have 250 ng per restriction digest reaction. Add 2 μL of buffer, 1 μL of enzyme, and fill to 20 μL with water. (Always add enzyme last). Then incubate at 37 ºC for 30 mins-1hr. | We want to have 250 ng per restriction digest reaction. Add 2 μL of buffer, 1 μL of enzyme, and fill to 20 μL with water. (Always add enzyme last). Then incubate at 37 ºC for 30 mins-1hr. | ||
</p> | </p> | ||
| − | + | <table style="width:45%"> | |
| + | <th>Sample</th> | ||
| + | <th>Amount of DNA</th> | ||
| + | <th>Amount of Cut Smart Buffer</th> | ||
| + | <th>Amount of Water</th> | ||
| + | <th>Amount of Enzyme (EcoRI)</th> | ||
| + | <tr> | ||
| + | <td>pBAD-D4</td> | ||
| + | <td>2.02 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>14.98 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 1:</td> | ||
| + | <td>2.57 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>14.43 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 2:</td> | ||
| + | <td>6.31 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>10.69 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 3:</td> | ||
| + | <td>5.29 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>11.71 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 4:</td> | ||
| + | <td>5.32 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>11.68 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 5:</td> | ||
| + | <td>5.98 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>11.02 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 6:</td> | ||
| + | <td>4.33 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>12.7 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 7:</td> | ||
| + | <td>7.44 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>9.56 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 8:</td> | ||
| + | <td>4.84 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>12.16 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 9:</td> | ||
| + | <td>6.02 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>10.98 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sample 10:</td> | ||
| + | <td>10.87 μL</td> | ||
| + | <td>2 μL</td> | ||
| + | <td>6.13 μL</td> | ||
| + | <td>1 μL</td> | ||
| + | </tr> | ||
| + | </table> | ||
</section> | </section> | ||
</div> | </div> | ||
Revision as of 20:48, 11 October 2018
Day 1
Miniprep bacteria with pBAD-D4 (name of the plasmid in which we will be inserting the Chitinase genes, tags, etc.) to isolate the pBAD backbone Final DNA concentration: 123.7 ng/μL
Day 2
Restriction digest using BamHI and EcoRI to check for bacterial transformation to check to make sure that the plasmid is the expected length (will also send samples for sequencing) Wells: 1. DNA Ladder. 2. Just EcoRI 3. Just BamHI. 4. No enzyme. 5. Both enzymes
Day 3
Made KPi Buffer… (used in Chitinase Assay)
- Prepare 800 mL of dH2O in a suitable container.
- Add 2.405 g of K2HPO4 to the solution.
- Add 11.73 g of KH2PO4 to the solution.
- Add distilled water until volume is 1 L.
Day 4
Started Cloning of pBAD-GST-ChiA-FLAG construct. (Function of GST and FLAG: these are protein tags (onto chitinase) to purify and detect chitinase respectively) Did the restriction digest portion, will do gel purification, ligation, and plating tomorrow For protocol click here
Day 5
Ran gel of restriction digest of pBAD only and did Gel Purification (very straightforward after PCR, you want only the copied DNA) of restriction digest of GST/gBlock as we want to preserve the amount of DNA gBlock that we have and will lose less sample via PCR purification. Final concentrations: pBAD: 22 ng/μL gBlock: 10.6 ng/μL Did DNA ligation and plated BL21 competent cells cloned with GST-ChiA full construct (complete protocol is linked in yesterday’s log)
Day 6
Selected 10 colonies from Vector+Insert plate and put in 4 mL of LB+Ampicillin (LB is nutrients for bacterial growth, Ampicillin assists selection of transformed bacteria) media. Incubate in 37 ºC shaker for 24 hrs. Also, did restriction digest and gel on pBAD-D4 vector using MluI and HindIII to check and make sure the enzymes are cutting properly. (If DNA length match expected length, then enzymes are working properly) There is a chance we will have to do ligation and stuff again because there aren’t that many colonies. We will most likely use primers to enhance the gBlock/insert DNA and then try again. Depending on how these colonies turn out after we sequence them. Vector+Insert colony:
Day 7
Did Miniprep of the 10 bacteria colonies that we selected from Wednesday. Used NanoDrop machine to find DNA concentrations of all 10 samples.