Team:Lethbridge/Notebook/Lab Work/July

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Back to Lab Work

Contents

July 2010

July 3/2010

(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.

July 5/2010

(In Lab: JV, AV, HB)

Objective: Run a 1% agarose gel of purified PCR samples from June 24/10

Method:

LaneSampleComponents (µL)
11kb Ladder0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
21 - pBAD (A4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
32 - pBAD (A5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
43 - SRBS (A6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
54 - SRBS (A7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
65 - CFP Complete (A8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
76 - SRBS (A10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
87 - EYFP (B1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
98 - N term tag (B2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
109 - pSB NEYFP (B4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1110 - pSB NEYFP (B5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1211 - CFP (B6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1312 - pBAD-TetR (B10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1413 - D31 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1514 - C term (D4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1615 - C term (D5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1716 - pLacI (D6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1817 - NEYFP (E2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1918 - CEYFP (E6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
2019 - CEYFP (E7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11kb ladder0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
220 - EYFP (E8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
321 - EYFP (E9)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
422 - EYFP (E10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
523 - ECFP (F1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
624 - ECFP (F2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
725 - ECFP (F3)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
826 - pBAD-TetR (F4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
927 - pBAD-TetR (F5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1028 - EYFP (G1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1129 - pSB CEYFP (G4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1230 - pBAD (1) (G6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1331 - pBAD (2) (G7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1432 - N term tag (G8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1533 - lumazine (G9)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O

Ran gel at 100V for 45 minutes.

Picture to come!!!!!!!!!

July 5/2010 Evening

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

July 6/2010

(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:

  • After checking the the sequencing it was evident that our promotor is always off. It is turned off by the product of the gene TetR. Which is not part of our construct.
Time (hours)OD (600λ)
00.071
10.390
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:

ComponentVolume (µL)
DNA0.071
Buffer Orange0.390
NotI0.606
MilliQ H2O1.250

Incubated for 1 hour at 37oC. Heat killed on heat block at 80oC for 20 mins.

2) 1% Agarose gel

LaneSampleVolume (µL)
11 kb Ladder0.5 Ladder + 2 Loading dye (6x) + 7.5 MilliQ H2O
2PET28a restricted8 DNA + 2 Loading dye (6x)
3PET28a8 DNA + 2 Loading dye (6x)
4Lumazine restricted8 DNA + 2 Loading dye (6x)
5Lumazine8 DNA + 2 Loading dye (6x)
6mms6 restricted8 DNA + 2 Loading dye (6x)
7mms68 DNA + 2 Loading dye (6x)
8xylE restricted8 DNA + 2 Loading dye (6x)
9xylE8 DNA + 2 Loading dye (6x)
10EmptyEmpty

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

July 8/2010

(In lab: JV, AV, HB, HS)
Objective:

Anthony does not understand the stuff written in the lab book.

July 8/2010 - Evening

(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:

PlateNumber of Colonies
50 µL TetR0
200 µL TetR5
50 µL pTetR4
200 µL pTetR34
50 µL pUC193
200 µL pUC194

July 9/2010

(In lab: JV)
Objective:To overexpress pLacI-mRBS-mms6-dT construct.

Method:Used the Overexpression Protocol.

Time (hours)OD (600λ)
00.052
10.111
20.315
2.50.449
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!!!!!!!!!!!

July 10/2010

(In lab: JV)
Objective:To determine if maxipreps finished on July 9th and 10th have significant concentrations of DNA.

Method:Run 1% Agarose gel

LaneSampleComponents (µL)
1dT2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
2mms6 (1)2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
3mms6 (2)2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
4pBAD-TetR (1)2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
5pBAD-TetR (2)2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
6mRBS2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
7pLacI2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
8sRBS2 DNA + 2 loading dye (6x) + 6 MilliQ H2O
91 kb Ladder0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H2O
10EmptyEmpty

Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes.

AGAROSE GEL PICTURE

July 12/2010

(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.

ComponentVolume (µL)
pDNA2
Red Buffer2
EcoRI0.25
SpeI0.25
MilliQ H2O15.5

2) Restrict "Part 2" BioBrick: dT with EcoRI and XbaI.

ComponentVolume (µL)2.5x Volume (µL)
pDNA25
Orange Buffer25
EcoRI0.250.625
XbaI0.250.625
MilliQ H2O15.538.75

3) 1% Agarose gel (1x TAE) of restricted DNA

LaneSampleComponent (µL)
11 kb Ladder0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H2O
2dT8 DNA + 2 loading dye (6x)
3dT restricted8 DNA + 2 loading dye (6x)
4mms68 DNA + 2 loading dye (6x)
5mms6 restricted8 DNA + 2 loading dye (6x)
6xylE8 DNA + 2 loading dye (6x)
7xylE restricted8 DNA + 2 loading dye (6x)
8Lumazine8 DNA + 2 loading dye (6x)
9Lumazine restricted8 DNA + 2 loading dye (6x)
10EmptyEmpty

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.

July 12/2010 - Evening

(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:

ComponentVolume (µL)
T4 DNA Ligase0.25
10x T4 Ligation Buffer2
dT8.3
mms69.2
MilliQ H2O0.25

dT to xylE:

ComponentVolume (µL)
T4 DNA Ligase0.25
10x T4 Ligation Buffer2
dT8.3
xylE3.4
MilliQ H2O6.05

dT to lumazine:

ComponentVolume (µL)
T4 DNA Ligase0.25
10x T4 Ligation Buffer2
dT8.3
lumazine3.35
MilliQ H2O6.1

Ligations were incubated at room temperature overnight.


Objective: Prepare mastermix for four PCR reactions for following day.

Method:

ComponentVolume (µL)5x Volume (µL)
10mM dNTPs15
5x Phusion Buffer420
Forward Primer (VF2)15
Reverse Primer (VR)15
MilliQ H2O10.854

July 13/2010

(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

LaneSampleComponents (µL)
150 bp Ladder1 ladder + 2 loading dye (6x) + 7 MilliQ H2O
2mms6-dT8 PCR product + 2 loading dye (6x)
3xylE-dT8 PCR product + 2 loading dye (6x)
4lumazine-dT8 PCR product + 2 loading dye (6x)
5dT8 PCR product + 2 loading dye (6x)
6EmptyEmpty
7EmptyEmpty
8EmptyEmpty
9EmptyEmpty
10EmptyEmpty

Ran at 100V for __ minutes. Stained in EtBr for 10 minutes.

AGAROSE GEL PICTURE

July 14/2010

(in lab:J.S, K.G )
Objective: Inoculate culture for maxi prep with placI

Method: To a 450mL solution of LB media 4.5µL of ampicillin was added with glycerol placI aseptically.

(in lab:J.S, K.G )
Objective: Restriction of dT and mms6

protocol

1)

ComponentVolume (µL)
Buffer&*2
10x T4 Milli Q H2O15.5
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:

ComponentVolume (µL)
T4 DNA Ligase0.25
10x T4 Ligation Buffer2
dT8.3
mms69.2
MilliQ H2O0.25

dT to xylE:

ComponentVolume (µL)
T4 DNA Ligase0.25
10x T4 Ligation Buffer2
dT8.3
xylE3.4
MilliQ H2O6.05

dT to lumazine:

ComponentVolume (µL)
T4 DNA Ligase0.25
10x T4 Ligation Buffer2
dT8.3
lumazine3.35
MilliQ H2O6.1

2) Incubated reaction overnight at room temperature

July 15/2010

(in lab: AV)
Objective: Maxiprep pLacI and mms6.

componentpallet weight (g)
mms61.02
pLacI1.54

July 15/2010 Evening

(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 coloniesComponents (µL)
1200µL xylE-dt July 120
2200µL mms6-dt July 121
3200µL lumazine-dt1
4200µL mms6 maxiprep July 6Lawn
5200µL positve control puC19 into BL21(DE3)Lawn
6200µL xylE-dt July 140
7200µL mms6-dt July 14120
8200µL lumazine-dt July140
9200µL mms6 maxiprep July 10.

*because of a shortage of plates not all transformations were plated at 50µL and 200µL

July 16,2010

(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

ComponentVolume (µL)
Milli-Q H2O<54
Pfu Buffer + MgSO420
10 mM dMTPs5
forward primer5
reverse primer5

89 µL TOTAL--> 17.8 into each PCR reaction

+ 2 µL ligation + 2 µL Pfu polymerase

Ran iGem-ligTest in thermocycler


July 19, 2010

(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

ComponentVolume (µL)
Milli-Q H2O15.5
Red Buffer42
SpeI0.25
EcoRI0.25
pDNA2

dT

ComponentVolume (µL)
Milli-Q H2O38.75
Orange Buffer45
XbaI0.625
EcoRI0.625
pDNA5

sRBS

ComponentVolume (µL)
Milli-Q H2O15.5
Red Buffer42
xBal0.25
EcoRI0.25
pDNA2

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

LaneSampleComponents (µL)
11kB Ladder0.5 ladder + 2 loading dye (5x) + 5.5 MilliQ H2O
2pLacI6 pDNA + 2 loading dye (5x)
3pLacI restriction digest6 pDNA + 2 loading dye (5x)
4sRBS6 pDNA + 2 loading dye (5x)
5sRBS restriction digest6 pDNA + 2 loading dye (5x)
6xylE6 pDNA + 2 loading dye (5x)
7xylE restriction digest6 pDNA + 2 loading dye (5x)
8lumazine6 pDNA + 2 loading dye (5x)
9lumazine restriction digest6 pDNA + 2 loading dye (5x)
10dT6 pDNA + 2 loading dye (5x)
11dT restriction digest6 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

LaneContent Quantity of DNA (ng/µL)
11kB Ladder12.5
3pLacI restriction digest5.21
5sRBS restriction digest3.72
7xylErestriction digest3.36
9Lumazine restriction digest2.63
11dT restriction digest2.98

July 19, 2010 Evening

(K.G)
Objective: Ligate together rbs-xylE and dT, lumazine and dT, also pLacI and sRBS.

Method: All ligation mixes had:

  • 10X T4 Ligation Buffer
  • Milli-Q H2O to fill to 20µL
  • T4 DNA Ligase
  • 20ng plasmid DNA

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.

July 20, 2010

(AV, HB)
Objective:Miniprep lumazine-dt and 4 N-terminus tags and analyze.

Method:

  • Use boiling lysis miniprep to isolate plasmid DNA.
  • PCR amplify BioBrick part to determine if the correct DNA was isolated.
  • Ran a 1% Agarose gel to visualize PCR products.

AGAROSE GEL PICTURE

July 20, 2010 Evening

(AV, HB)
Objective:Transform ligation done on July 19, 2010 in to competent DH5α cells.

  • xylE-dT
  • lumazine synthase-dT
  • pLacI-sRBS
  • EYFP and ECFP

Method:

Results:

  • xylE-dT no colonies
  • pLacI-sRBS
  • EYFP
  • lumazine-dT
  • ECFP

July 21, 2010

(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

July 21, 2010 Evening

(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:.

  • K249001
  • K249008
  • K249004
  • mms6-dt
  • lumazine-dt
  • K249017
  • K249014
  • K249005
  • ECFP
  • EYFP
  • K249006
  • N-term tag
  • xylE-dt
  • SRBS-pLacI
  • dt
  • pTet maxiprep
  • pET-28(a) maxiprep
  • TetR maxiprep

Gel ran at 100V for 60minutes.
Results: The PCR's did not work.

July 27, 2010

(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:

  • dT (control)
  • E0020 (ECFP)
  • E0030 (EYFP)
  • K249001
  • K249004
  • K249005
  • K249006
  • K249008
  • K249014
  • K249017

The following parts were miniprep'd using Qiagen Miniprep Kit:

  • K249005
  • K249008

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.

July 28, 2010

(in lab: JV)

Objective: To create a large quantity of pSB1C3 plasmid.

Method: PCR amplified pSB1C3 received from iGEM headquarters.

July 29, 2010

(in lab: HB, AV)

Objective: To PCR amplify the minipreps on July 27, 2010.

Method:
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