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
Analyze sequence of ER, design new primer for continued sequencing
Plan experiments using pNT001 and pNT002
Results
Estrogen receptor (K123003) sequencing is back. Again, the translation is right, but the bases don't match. The stop codon is not in this read. We will keep sequencing.
Concentration of Purified PCR products from June 28 and June 29
| Tube Number |
Concentration(ng/ul) |
| 6/28 3 |
15.2 |
| 6/28 4 |
49.1 |
| 6/28 5 |
42.0 |
| 6/28 6 |
112.0 |
| 6/29 1 |
112.7 |
| 6/29 2 |
168.0 |
June 30
Miniprep 5 K145001 cultures and send them off for sequencing before noon
Make glycerol stocks of mCherry (J06702), Lac Promoter (R0010), double terminator (B0014 and B0015) and Tet Promoter (R0040)
Ask Joe if we need to PCR plasmid or if he has linear plasmid
If we need to linearize: Borrow plasmid primers and plasmid, do PCR. Repeat failed PCR (Primers 9 and 10 and DNA B0014) along with it
If we don't, then repeat failed PCR
Get recipe from Joe for gibson mix and gibson protocol, make gibson mix, and run gibson reaction and transform
For gels tomorrow: Use Joe's sybr safe.
This Week
Email other teams
Use pNT001 and pNT002 to test degradation parts
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