June 26
Start overnight cultures of T7 polymerase (K145001), mCherry (J06702), Lac Promoter (R0010), double terminator (B0014 and B0015) and Tet Promoter (R0040)
June 27
Miniprep of T7 polymerase (K145001), mCherry (J06702), Lac Promoter (R0010), double terminator (B0014 and B0015) and Tet Promoter (R0040) and submit for sequencing
Transfer 0.5 mL aliquots of BPA and 5 mL aliquots 17a-ethynylestradiol cultures from June 23 to fresh minimal media
Checked DDT and nonylphenol cultures, will wait until tomorrow to transfer
Ethanol precipitation of DNA extractions from LA river samples following protocol
Streak out cells from fosmid kit
Prepare antibiotic stocks (Ampicillin, Chloramphenicol)
Results
Miniprep
| Part |
Concentration(ng/ul) |
| B0014 |
230.5 |
| B0015 |
254.1 |
| J06702 |
301.3 |
| K145001 |
205.6 |
| R0010 |
117.3 |
| R0040 |
156.8 |
We found growth in one of our BPA enrichment cultures, and slight growth in our ethinyl estradiol cultures. We transferred these vials today.
June 28
Send off continued forward sequencing for HER
PCR for Gibson assembly of PNT001 and PNT002
Gel and PCR purification of PCR products
Analysis of sequencing results from yesterday
Transfer DDT and nonylphenol cultures to new media
Transform mCherry (J06702), Lac Promoter (R0010), double terminator (B0014 and B0015) and Tet Promoter (R0040)for creation of glycerol stocks
Results
Sequencing: All biobricks showed correct sequence except T7 Polymerase
Ran a gel of the PCR products. Strangely, lanes 3 and 4 show no DNA. We will repeat the PCR of lanes 3-5 and Gibson assemble pNT001 (R0010, K123000, B0014) later in the week.
EtOH precipitation of Soil Extractions from June 21
| Tube/Location Number |
Concentration(ng/ul) |
| 1 |
13.6 |
| 2 |
15.2 |
| 4 |
8.3 |
| 5 |
11.4 |
| 6 |
25.5 |
| 7 |
23.5 |
| 9 |
261.3 |
| 10 |
74.0 |
June 29
Redo PCR of R0010 and K123000
Gibson assemble pNT002, pNT001 if redo of PCR works
Order primers and vectors for 16s sequencing
Analyze sequence of ER, design test plasmids if sequence of ER is correct, if not, plan repair
Plan experiments using pNT001 and pNT002
This Week
Email other teams
Use pNT001 and pNT002 to test degradation parts
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