IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week1/Chemical and Light: Difference between revisions
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===Restriction Digest=== | ===Restriction Digest=== | ||
06/24: Digest of P17 DNA with Xba1 and Spe1. The entire plasmid is 4425 bp and the short fragment (containing only the BioBrick) is 902 bp. | 06/24: Digest of P17 DNA with Xba1 and Spe1. The entire plasmid is 4425 bp and the short fragment (containing only the BioBrick) is 902 bp. | ||
15 μl DNA | 15 μl DNA | ||
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1 μl Spe1 | 1 μl Spe1 | ||
Incubate overnight at 37 °C | Incubate overnight at 37 °C |
Revision as of 10:24, 25 June 2008
Chemical
Results from Friday (6/20) Electroporation and Transformation in S1
6/23:
Name Registry Name Description Origin Marker Observations P1a J23113 Low promoter GFP tester pMB1 Kan 16 pink colonies of approx 7mm diameter P2a J23150 Medium promoter GFP tester pMB1 Kan no growth P3a J23151 High promoter GFP tester pMB1 Kan 8 pink colonies of approx 7mm diameter P12 pETDuet-1 Low copy plasmid pBR322-derived ColE1 Amp Lawn P13 pCDFDuet-1 Low copy plasmid CloDF13 Sm 41 pink colonies and some small yellow ones that may be E. coli P14 pCOLADuet-1 Low copy plasmid ColA Kan No growth
Growing cultures of E. coli and S1
6/23:
Picked colonies of E. coli and S1 and grew overnight at 37 and 30 degrees, respectively.
Strain Plasmid Name Registry Name Plate Information Marker Colonies Picked E. coli P15 A1 6/18 electrocompetent Cm 2 TOP10 P4 pACYC duet 6/19 restreak electrocomp Cm 2 TOP10 P7 pSB1A2 6/19 restreak electrocomp big and small Cm 2 (1 from big, 1 from small) TOP10 P8 BBa_J04450 6/19 restreak electrocomp big and small Amp 2 (1 from big, 1 from small) TOP10 P9 BBa_J04430 6/19 restreak electrocomp big and small Amp 2 (1 from big, 1 from small) TOP10 P10 BBa_I715038 6/19 restreak electrocomp big and small Amp 2 (1 from big, 1 from small) S1 P4 pACYC duet 6/18 electrocomp Cm 2 S1 P21 A1 6/19 electrocomp Cm 2 S1 P13 pCDFDuet-1 6/20 electrocomp Sm 2 S1 P1a J23113 6/20 electrocomp Kan 3 S1 P3a J23151 6/20 electrocomp Kan 3
Total number of cultures: 24
6/24:
None of the samples in the LB amp media grew. All the others grew and then were miniprepped and stored in glycerol.
- Glycerol stocks: 250 uL of culture in 500 uL of 20% LB glycerol.
Concentrations of DNA from minipreps:
Plasmid Organism Concentration (ng/uL) P1A a S1 92.54 P1A b S1 55.34 P1A c S1 58.21 P3A a S1 62.43 P3A b S1 168.41 P3A c S1 151.49 P4 S1 150.32 P13 S1 435.39 P21 S1 219.33 P4A a TOP10 49.81 P4A b TOP10 47.22 P21A a TOP10 61.16 P21A b TOP10 55.71
Transformation of P5 and P6 into E3
6/24:
P5 and P6 were punched from the book and transformed in E3 (the death resistant strain)
16S PCR to Identify S1 and E. Coli
6/24:
Performed PCR as per protocol listed in General Protocols.
Primer Sets
- 1 = Shewanella F and Shewanella R
- 2 = E. coli F and E. coli R
- 3 = Common F and Common R
Tube # Strain/ Colony of Template DNA Primer Set Used Volume of Template DNA Volume of Primer Volume of Supermix Volume of Water 1 S1 P1a 1 (SF & SR) 1uL 1uL (each) 45uL 2uL 2 S1 P1a 1 (SF & SR) 3uL 1uL (each) 45uL 2uL 3 S1 P1a 2 (EF & ER) 1uL 1uL (each) 45uL 2uL 4 S1 P1a 2 (EF & ER) 3uL 1uL (each) 45uL 2uL 5 S1 P1a 3 (CF & CR) 1uL 1uL (each) 45uL 2uL 6 S1 P1a 3 (CF & CR) 3uL 1uL (each) 45uL 2uL 7 S1 P3a 1 (SF & SR) 1uL 1uL (each) 45uL 2uL 8 S1 P3a 1 (SF & SR) 3uL 1uL (each) 45uL 2uL 9 S1 P3a 2 (EF & ER) 1uL 1uL (each) 45uL 2uL 10 S1 P3a 2 (EF & ER) 3uL 1uL (each) 45uL 2uL 11 S1 P3a 3 (CF & CR) 1uL 1uL (each) 45uL 2uL 12 S1 P3a 3 (CF & CR) 3uL 1uL (each) 45uL 2uL 13 S1 MR-1 1 (SF & SR) 1uL 1uL (each) 45uL 2uL 14 S1 MR-1 1 (SF & SR) 3uL 1uL (each) 45uL 2uL 15 S1 MR-1 2 (EF & ER) 1uL 1uL (each) 45uL 2uL 16 S1 MR-1 2 (EF & ER) 3uL 1uL (each) 45uL 2uL 17 S1 MR-1 3 (CF & CR) 1uL 1uL (each) 45uL 2uL 18 S1 MR-1 3 (CF & CR) 3uL 1uL (each) 45uL 2uL 19 E. coli P1a 1 (SF & SR) 1uL 1uL (each) 45uL 2uL 20 E. coli P1a 1 (SF & SR) 3uL 1uL (each) 45uL 2uL 21 E. coli P1a 2 (EF & ER) 1uL 1uL (each) 45uL 2uL 22 E. coli P1a 2 (EF & ER) 3uL 1uL (each) 45uL 2uL 23 E. coli P1a 3 (CF & CR) 1uL 1uL (each) 45uL 2uL 24 E. coli P1a 3 (CF & CR) 3uL 1uL (each) 45uL 2uL
6/25
Gel results from PCR:
Key:
Well # Strain/ Colony Primer Set Used Volume of Template DNA 1 1 Kb ladder N/A N/A 2 S1 P3a 1 (SF & SR) 3uL 3 S1 P3a 1 (SF & SR) 1uL 4 S1 P3a 2* (EF & ER) 1uL 5 S1 P3a 3 (CF & CR) 1uL 6 S1 P3a 3 (CF & CR) 3uL 7 S1 P1a 1 (SF & SR) 3uL 8 S1 P1a 1 (SF & SR) 1uL 9 S1 P1a 2 (EF & ER) 1uL 10 S1 P1a 2 (EF & ER) 3uL 11 S1 P1a 3 (CF & CR) 3uL 12 S1 P1a 3 (CF & CR) 1uL
Note: S1 P3a With Primer Set 2 and 3 uL of template DNA evaporated during PCR so was not included in the gel.
Key:
Well # Strain/ Colony Primer Set Used Volume of Template DNA 1 1 Kb ladder N/A N/A 2 S1 MR-1 1 (SF & SR) 1uL 3 S1 MR-1 2 (EF & ER) 1uL 4 S1 MR-1 3 (CF & CR) 1uL 5 100 bp Ladder N/A N/A 6 E.coli P1a 1 (SF & SR) 1uL 7 E.coli P1a 1 (SF & SR) 3uL 8 E.coli P1a 2 (EF & ER) 1uL 9 E.coli P1a 2 (EF & ER) 3uL 10* E.coli P1a 3 (CF & CR) 1uL 11 E.coli P1a 3 (CF & CR) 3uL 12 1 Kb Ladder N/A N/A
Note: There was a problem with the gel in Well 10, which most likely accounts for the lighter band.
Light
Transformations of P11, P15-20
Colony counts
6/23:
Name | Description | Origin | Size | Marker | Number of Colonies |
P11 | Light responsive system, dual regulation | pUC19-derived pMB1 | 4333 | Amp/Cm | 224 |
P15 | GFP with Tet promoter | ColE1 | 937 | Amp | 360 |
P16 | Tet repressible promoter | pMB1 | 54 | Amp | 264 |
P17 | Inverter (TetR w/o promoter and Tet promoter) | OriS, P1 lytic, F1 | 902 | Kan | 2 |
P18 | lambda promoter (cI regulated) | pMB1 | 49 | Amp | 112 |
P19 | RFP with lambda promoter | pMB1 | 918 | Amp | 104 |
P20 | GFP (LVA tagged) with Lac promoter | pMB1 | 1122 | Amp, Kan | 136 |
Restreaking
06/23: P16, which has GFP under a constitutive Tet promoter did not appear to fluoresce (none of the fluorescent constructs did), so we restreaked all the of the plates (with the same antibiotic on which they were originally grown). They were left in the 37 degree incubator along with a blank Kan and a blank Amp plate (- controls).
06/24: All of the restreaked plates grew and were streaked to individual colonies. Both the Kan and Amp negative controls (no cells) were blank. However, p15 (GFP under pTet) did not appear to glow.
Overnight cultures
06/23: The above was repeated in 5mL LB liquid cultures instead of plates.
06/24: None of the cultures in LB Amp grew (E1P17 in LB Kan did grow). We recultured the other samples in LB Amp and made a master plate with patches from the same colonies that were cultured. Since others seemed to get similar problems (no growth in liquid culture but growth on plates for Amp), we made fresh LB Amp (from freshly made Amp stock) and repeated the cultures.
06/25: These cultures did not grow in the new medium either. We plated them on new LB amp plates (new plates 1X amp or 1/1000) and cultured them in low concentration LB amp (0.5X or 1/2000 amp).
Miniprep
06/24: Miniprep of E1P17. The DNA is in the -20 °C iGEM freezer. The DNA concentration is 54.8 ng/μL.
Restriction Digest
06/24: Digest of P17 DNA with Xba1 and Spe1. The entire plasmid is 4425 bp and the short fragment (containing only the BioBrick) is 902 bp.
15 μl DNA
6.25 μl water
2.5 μl NEB Buffer 2
0.25 μl 100X BSA
1 μl Xba1
1 μl Spe1
Incubate overnight at 37 °C
06/25: Since the previous digest was unsuccessful, we digested the same plasmid with different combinations of restriction enzymes. We also tested two other plasmids from the chemical group (P4 and P21).
1. P17 with XbaI, SpeI
2. P17 with EcoRI, SpeI
3. P17 with EcoRI, PstI
4. P17 with XbaI, PstI
5. P17 with XbaI
6. P17 with SpeI
7. P4 with XbaI, SpeI
8. P4 with EcoRI, SpeI
9. P20 with XbaI, PstI
10. P20 with EcoRI, PstI
Transformations of P22-24
06/24: Transformations occurred in duplicate (TOP10 and DH5α) with Amp selection. 06/25: All plates had colonies. We inoculated liquid cultures of cells with each of these plasmids in low (0.5X or 1/2000 amp) and high (1X or 1/1000 amp) concentration LB amp.
More transformations
06/25: Transformed P17, P20, P25, and P26 in DH5α cells. P17 and P20 have Kan resistance; P25 and P26 have Amp resistance.