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(In Lab: JS)
Objective: To restrict and ligate pBAD-TetR and YEPs into pSB1C3 backbone.
Method: Used Restriction of Plasmid DNA protocol and ligated the parts into pSB1C3.
(In Lab: JV, AV, HB)
Objective: Run a 1% agarose gel of purified PCR samples from June 24/10
Method:
Lane | Sample | Components (µL) |
1 | 1kb Ladder | 0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O |
2 | 1 - pBAD (A4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
3 | 2 - pBAD (A5) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
4 | 3 - SRBS (A6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
5 | 4 - SRBS (A7) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
6 | 5 - CFP Complete (A8) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
7 | 6 - SRBS (A10) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
8 | 7 - EYFP (B1) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
9 | 8 - N term tag (B2) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
10 | 9 - pSB NEYFP (B4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
11 | 10 - pSB NEYFP (B5) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
12 | 11 - CFP (B6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
13 | 12 - pBAD-TetR (B10) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
14 | 13 - D3 | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
15 | 14 - C term (D4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
16 | 15 - C term (D5) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
17 | 16 - pLacI (D6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
18 | 17 - NEYFP (E2) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
19 | 18 - CEYFP (E6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
20 | 19 - CEYFP (E7) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
1 | 1kb ladder | 0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O |
2 | 20 - EYFP (E8) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
3 | 21 - EYFP (E9) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
4 | 22 - EYFP (E10) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
5 | 23 - ECFP (F1) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
6 | 24 - ECFP (F2) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
7 | 25 - ECFP (F3) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
8 | 26 - pBAD-TetR (F4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
9 | 27 - pBAD-TetR (F5) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
10 | 28 - EYFP (G1) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
11 | 29 - pSB CEYFP (G4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
12 | 30 - pBAD (1) (G6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
13 | 31 - pBAD (2) (G7) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
14 | 32 - N term tag (G8) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
15 | 33 - lumazine (G9) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
Ran gel at 100V for 45 minutes.
Picture to come!!!!!!!!!
Objective: To over-express CFP complete in DH5α
Method:
1) Inoculated 6mL culture with ampicillin and glycerol stock A6-May 13,2010 CFP complete 2) Went in the shaker at 6:50pm
(In lab: JV, AV, HB)
Objective: To continue the over-expression of CFP complete in DH5α
Method:
1) Put both cultures (taken out of shaker at 9:15am) and put them in 500mL of LB w/ Amp.
2) initial OD was 0.071 (600λ)
Issue:
Time (hours) | OD (600λ) |
0 | 0.071 |
1 | 0.390 |
1.5(T0) | 0.606 |
2.5 (T1) | 1.250 |
3.5(T2) | 3.04 |
4.5(T3) | 2.75 |
Results: Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.
Objective: To determine if maxipreps were successful via restriction by NotI and running on 1% Agarose gel
Method:
1) Restriction:
Component | Volume (µL) |
DNA | 0.071 |
Buffer Orange | 0.390 |
NotI | 0.606 |
MilliQ H2O | 1.250 |
Incubated for 1 hour at 37oC.
Heat killed on heat block at 80oC for 20 mins.
2) 1% Agarose gel
Lane | Sample | Volume (µL) |
1 | 1 kb Ladder | 0.5 Ladder + 2 Loading dye (6x) + 7.5 MilliQ H2O |
2 | PET28a restricted | 8 DNA + 2 Loading dye (6x) |
3 | PET28a | 8 DNA + 2 Loading dye (6x) |
4 | Lumazine restricted | 8 DNA + 2 Loading dye (6x) |
5 | Lumazine | 8 DNA + 2 Loading dye (6x) |
6 | mms6 restricted | 8 DNA + 2 Loading dye (6x) |
7 | mms6 | 8 DNA + 2 Loading dye (6x) |
8 | xylE restricted | 8 DNA + 2 Loading dye (6x) |
9 | xylE | 8 DNA + 2 Loading dye (6x) |
10 | Empty | Empty |
Ran at 100V for 80 mins.
Picture to come!!!!!!!!!
More to fill in, but Anthony does not understand the stuff written in the lab book
(In lab: JV, AV, HB, HS)
Objective:
Anthony does not understand the stuff written in the lab book.
(In lab: KG)
Objective:To transform TetR in pSB1A2 plasmid (BBa_C0040 - 2010 iGEM Distribution Kit Plate Well 4A) and pTetR in pSB1A2 plasmid (BBa_R0040 - 2010 iGEM Distribution Kit Plate Well 6) into DH5α.
Method:Used the Competent Cell Transformation Protocol as well as transformed pUC19 as a positive control.
Results:
Plate | Number of Colonies |
50 µL TetR | 0 |
200 µL TetR | 5 |
50 µL pTetR | 4 |
200 µL pTetR | 34 |
50 µL pUC19 | 3 |
200 µL pUC19 | 4 |
(In lab: JV)
Objective:To overexpress pLacI-mRBS-mms6-dT construct.
Method:Used the Overexpression Protocol.
Time (hours) | OD (600λ) |
0 | 0.052 |
1 | 0.111 |
2 | 0.315 |
2.5 | 0.449 |
3(T0) | 0.772 |
4(T1) | 2.22 |
5(T2) | 2.06 |
6(T3) | 2.50 |
Samples were run on a 15% SDS PAGE for 20 minutes at 80V and 1 hour at 200V.
SDS PAGE picture!!!!!!!!!!!
(In lab: JV)
Objective:To determine if maxipreps finished on July 9th and 10th have significant concentrations of DNA.
Method:Run 1% Agarose gel
Lane | Sample | Components (µL) |
1 | dT | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
2 | mms6 (1) | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
3 | mms6 (2) | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
4 | pBAD-TetR (1) | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
5 | pBAD-TetR (2) | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
6 | mRBS | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
7 | pLacI | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
8 | sRBS | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
9 | 1 kb Ladder | 0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H2O |
10 | Empty | Empty |
Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes.
AGAROSE GEL PICTURE
(In lab: AV,HB,JV)
Objective: Maxiprep pLacI (A2) from glycerol stock.
Method: Used Maxiprep Protocol. Cell pellet weighed 1.02g.
Objective: Add dT to the end of mms6, xylE, and lumazine.
Method:
1) Restrict "Part 1" BioBricks: mms6, xylE, and lumazine with EcoRI and SpeI.
Component | Volume (µL) |
pDNA | 2 |
Red Buffer | 2 |
EcoRI | 0.25 |
SpeI | 0.25 |
MilliQ H2O | 15.5 |
2) Restrict "Part 2" BioBrick: dT with EcoRI and XbaI.
Component | Volume (µL) | 2.5x Volume (µL) |
pDNA | 2 | 5 |
Orange Buffer | 2 | 5 |
EcoRI | 0.25 | 0.625 |
XbaI | 0.25 | 0.625 |
MilliQ H2O | 15.5 | 38.75 |
3) 1% Agarose gel (1x TAE) of restricted DNA
Lane | Sample | Component (µL) |
1 | 1 kb Ladder | 0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H2O |
2 | dT | 8 DNA + 2 loading dye (6x) |
3 | dT restricted | 8 DNA + 2 loading dye (6x) |
4 | mms6 | 8 DNA + 2 loading dye (6x) |
5 | mms6 restricted | 8 DNA + 2 loading dye (6x) |
6 | xylE | 8 DNA + 2 loading dye (6x) |
7 | xylE restricted | 8 DNA + 2 loading dye (6x) |
8 | Lumazine | 8 DNA + 2 loading dye (6x) |
9 | Lumazine restricted | 8 DNA + 2 loading dye (6x) |
10 | Empty | Empty |
Ran at 100V for 43 minutes. Stained in EtBr for 10 minutes.
AGAROSE GEL PICTURE
4) Ligate restricted dT to the ends of the "Part 1" Biobricks.
(In lab: KG,TF)
Objective: Continue with addition of dT to the ends of mms6, xylE, and lumazine.
Method:
4) Ligate restricted dT to the ends of the "Part 1" Biobricks.
dT to mms6:
Component | Volume (µL) |
T4 DNA Ligase | 0.25 |
10x T4 Ligation Buffer | 2 |
dT | 8.3 |
mms6 | 9.2 |
MilliQ H2O | 0.25 |
dT to xylE:
Component | Volume (µL) |
T4 DNA Ligase | 0.25 |
10x T4 Ligation Buffer | 2 |
dT | 8.3 |
xylE | 3.4 |
MilliQ H2O | 6.05 |
dT to lumazine:
Component | Volume (µL) |
T4 DNA Ligase | 0.25 |
10x T4 Ligation Buffer | 2 |
dT | 8.3 |
lumazine | 3.35 |
MilliQ H2O | 6.1 |
Ligations were incubated at room temperature overnight.
Objective: Prepare mastermix for four PCR reactions for following day.
Method:
Component | Volume (µL) | 5x Volume (µL) |
10mM dNTPs | 1 | 5 |
5x Phusion Buffer | 4 | 20 |
Forward Primer (VF2) | 1 | 5 |
Reverse Primer (VR) | 1 | 5 |
MilliQ H2O | 10.8 | 54 |
(in lab: JV)
Objective: To determine if ligations of previous day (July 12/2010) were successful.
Method:
1) Use prepared mastermix to run a PCR of ligated mms6-dT, ligated xylE-dT, ligated lumazine-dT, and control-dT
- To each PCR tube, add 17.8&mirco;L of mastermix, 0.2µL of Phusion DNA polymerase, and 2µL of template DNA.
- Ran PCR's for 36 cycles using the iGEM preset.
2) 2% Agarose gel
Lane | Sample | Components (µL) |
1 | 50 bp Ladder | 1 ladder + 2 loading dye (6x) + 7 MilliQ H2O |
2 | mms6-dT | 8 PCR product + 2 loading dye (6x) |
3 | xylE-dT | 8 PCR product + 2 loading dye (6x) |
4 | lumazine-dT | 8 PCR product + 2 loading dye (6x) |
5 | dT | 8 PCR product + 2 loading dye (6x) |
6 | Empty | Empty |
7 | Empty | Empty |
8 | Empty | Empty |
9 | Empty | Empty |
10 | Empty | Empty |
Ran at 100V for __ minutes. Stained in EtBr for 10 minutes.
AGAROSE GEL PICTURE
(in lab:J.S, K.G )
Objective: Inoculate culture for maxi prep with placI
Method: To a 450mL solution of LB media 4.5(µL) of ampicillin was added with glycerol placI aseptically.
(in lab:J.S, K.G )
Objective: Restriction of dT and mms6
protocol
1)
Component | Volume (µL) |
Buffer&* | 2 |
10x T4 Milli Q H2O | 15.5 |
pDNA** | 2 |
Restriction enzymes*** | 0.25 |
* Orange buffer was used for dT, and Red buffer was used for mms6
**pDNA was dT and mms6
***Restriction enzymes for dT were XbalI and EcoRI, and for mms6 SpeI and EcoRI
2) Reaction was incubated at 370C for 1 hour. Start 8:50pm till 9:50pm After incubation reaction was heat shocked at 800C for 20 minutes
(in lab:J.S, K.G )
Objective: Ligation of dT to each of mms6, xylE and lumazine.
protocol
1)
dT to mms6:
Component | Volume (µL) |
T4 DNA Ligase | 0.25 |
10x T4 Ligation Buffer | 2 |
dT | 8.3 |
mms6 | 9.2 |
MilliQ H2O | 0.25 |
dT to xylE:
Component | Volume (µL) |
T4 DNA Ligase | 0.25 |
10x T4 Ligation Buffer | 2 |
dT | 8.3 |
xylE | 3.4 |
MilliQ H2O | 6.05 |
dT to lumazine:
Component | Volume (µL) |
T4 DNA Ligase | 0.25 |
10x T4 Ligation Buffer | 2 |
dT | 8.3 |
lumazine | 3.35 |
MilliQ H2O | 6.1 |
2)
Incubated reaction overnight at room temperature
(in lab: AV)
Objective: Maxiprep pLacI and mms6.
component | pallet weight (g) |
mms6 | 1.02 |
pLacI | 1.54 |
(in lab: AV)
Objective: transform ligations from July 14 and July 12,2010;xylE/dt, mms6/dt lumazine/dt into DH5&alpha.
Also to transform mms6 from July 6 and July 10 into Bl21(DE3)
Protocol: to transform competent cells see protocol:[1]
* cells were incubated on ice for 30 minutes started at 7:00-7:30. **incubated for 1 hour from 8:00 till 9:00
Results | ||
plate | # of colonies | Components (µL) |
1 | 200µL xylE-dt July 12 | 0 |
2 | 200µL mms6-dt July 12 | 1 |
3 | 200µL lumazine-dt | 1 |
4 | 200µL mms6 maxiprep July 6 | Lawn |
5 | 200µL positve control puC19 into BL21(DE3) | Lawn |
6 | 200µL xylE-dt July 14 | 0 |
7 | 200µL mms6-dt July 14 | 120 |
8 | 200µL lumazine-dt July14 | 0 |
9 | 200µL mms6 maxiprep July 10 | . |
*because of a shortage of plates not all transformations were plated at 50µL and 200µL
(in lab: M.C, D.M)
Objective: PCR amplify mms6-dT, xylE-dT, lumazine-dT legations along with dT maxi prep for comparison
Protocol:
Master Mix
Component | Volume (µL) |
Milli-Q H2O< | 54 |
Pfu Buffer + MgSO4 | 20 |
10 mM dMTPs | 5 |
forward primer | 5 |
reverse primer | 5 |
89 µL TOTAL--> 17.8 into each PCR reaction
+ 2 µL ligation + 2 µL Pfu polymerase
Ran iGem-ligTest in thermocycler
(in lab: A.V, H.B)
Objective: Purification pf pLacI and mms6 maxipreps done on July 14 and 16, 2010 using the biobasic protocol for purification of PCR products.
Objective: Add pLacI to sRBS and add dT to xylE and lumazine
Method:
Restrict pLacI, xylE and lumazine with SpeI and EcoRI and restrict dT and sRBS with EcorI and XbaI
1)Restrictions:
pLacI, xylE, and Lumazine
Component | Volume (µL) |
Milli-Q H2O | 15.5 |
Red Buffer4 | 2 |
SpeI | 0.25 |
EcoRI | 0.25 |
pDNA | 2 |
dT
Component | Volume (µL) |
Milli-Q H2O | 38.75 |
Orange Buffer4 | 5 |
XbaI | 0.625 |
EcoRI | 0.625 |
pDNA | 5 |
sRBS
Component | Volume (µL) |
Milli-Q H2O | 15.5 |
Red Buffer4 | 2 |
xBal | 0.25 |
EcoRI | 0.25 |
pDNA | 2 |
Restriction incubation at 37.50C started at 11:55am. Ended at 12:55pm Heat killed enzymes at 800C for 20 minutes
2)Restrictions were run on a 1% agarose gel (1 X TAE)
1% Agarose gel
Lane | Sample | Components (µL) |
1 | 1kB Ladder | 0.5 ladder + 2 loading dye (5x) + 5.5 MilliQ H2O |
2 | pLacI | 6 pDNA + 2 loading dye (5x) |
3 | pLacI restriction digest | 6 pDNA + 2 loading dye (5x) |
4 | sRBS | 6 pDNA + 2 loading dye (5x) |
5 | sRBS restriction digest | 6 pDNA + 2 loading dye (5x) |
6 | xylE | 6 pDNA + 2 loading dye (5x) |
7 | xylE restriction digest | 6 pDNA + 2 loading dye (5x) |
8 | lumazine | 6 pDNA + 2 loading dye (5x) |
9 | lumazine restriction digest | 6 pDNA + 2 loading dye (5x) |
10 | dT | 6 pDNA + 2 loading dye (5x) |
11 | dT restriction digest | 6 pDNA + 2 loading dye (5x) |
Gel ran for 70 minutes at 100V and was stained in EtBr for 10 minutes
AGAROSE GEL PICTURE
Results after quantifying restriction digest
Lane | Content | Quantity of DNA (ng/µL) |
1 | 1kB Ladder | 12.5 |
3 | pLacI restriction digest | 5.21 |
5 | sRBS restriction digest | 3.72 |
7 | xylErestriction digest | 3.36 |
9 | Lumazine restriction digest | 2.63 |
11 | dT restriction digest | 2.98 |
(K.G)
Objective: Ligate together rbs-xylE and dT, lumazine and dT, also pLacI and sRBS.
Method: All ligation mixes had:
Ligations were left over-night at room temperature.
(J.V.)
Objective:Determine the results of the transformations done by K.G. on July 15/2010.
Method: Inoculate 5mL LB media w/Amp and colony. Incubated over night at 37oC.
Results: Lumazine Synthase with dT ligated onto it grew.
(AV, HB)
Objective:Miniprep lumazine-dt and 4 N-terminus tags and analyze.
Method:
AGAROSE GEL PICTURE
(AV, HB)
Objective:Transform ligation done on July 19, 2010 in to competent DH5α cells.
Method:
Results:
(JV)
Objective: Isolate plasmid DNA from pTet, TetR, pET-28(a).
Method:
(JV)
Objective: To induce over-expression of pLacI-RBS-Mms6-dt in BL21(DE3) cells.
Method: Used Overexpression.
Ran SDS gel for 78 minutes at 200V
(TF, AS)
Objective: Colony PCR to test for insertions of BioBrick construction, and for quality control of parts received from the Registry. Also to prepare DNA to be sent away for sequencing.
Method:
-Ran PCR's
-Ran 2% Agarose gel for:.
Gel ran at 100V for 60minutes.
Results: The PCR's did not work.
(JV)
Objective: Insert xylE, Mms6, and lumazine synthase into pET-28(a).
Method: A restriction digest was performed on xylE and Mms6 and ran for 90 minutes at 37 C
(in lab: JV, AV, HB)
Objective: To miniprep the overnight cultures of the parts ordered from the Parts Registry and to test the efficiency of the Qiagen Miniprep Kit.
Method:
The following parts were miniprep'd using Boiling Lysis Plasmid Preparation:
The following parts were miniprep'd using Qiagen Miniprep Kit:
Results: Qiagen Miniprep Kit produced a comparable quantity of DNA to Boiling Lysis Plasmid Preparation. Since the Qiagen Miniprep Kit is faster and easier, all minipreps will be done using the Qiagen Miniprep Kit.
(in lab: JV)
Objective: To create a large quantity of pSB1C3 plasmid.
Method: PCR amplified pSB1C3 received from iGEM headquarters.
(in lab: HB, AV)
Objective: To PCR amplify the minipreps on July 27, 2010.
Method: PCR amplified the 11 minipreps and dT control.
Results: PCR amplification was successful and 0.2 µL of polymerase will be used per PCR reaction in the future.
Objective: To maxiprep EYFP (E0030), ECFP (E0020), and Lumazine Synthase (K249002).
Method: Used Maxiprep
Cell Pellet | Weight (g) |
ECFP | 2.32 |
EYFP | 2.00 |
Lumazine Synthase | 2.66 |
(in lab: KG)
Objective: Run PCR products from the morning on a 2% agarose gel.
Method: Ran 2% agarose gel of the 11 minipreps and dT control and stained in EtBr for 17 minutes.
Results: AGAROSE GEL PICTURE