Contents 1 August 1.1 August 3, 2010 1.2 August 3, 2010 Evening 1.3 August 4, 2010 1.4 August 6/2010 Evening 1.5 Aug 9/2010 1.6 Aug 9/2010 Evening 1.7 Aug 10/2010

August

August 3, 2010

(in Lab: HB, AV, JV)

Objective: To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used Restriction of Plasmid DNA protocol.

• A front verctor was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes
• pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes
• A back vector was made in sRBS and mRBS plasmids with PstI and SpeI
• pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI
  

Construct	pDNA	buffer	Enzymes
pBAD-sRBS/mRBS	pBAD	Red	EcoRI and SpeI
pBAD-sRBS/mRBS	sRBS	Orange	XbaI and EcoRI
pBAD-sRBS/mRBS	mRBS	Orange	XbaI and EcoRII
sRBS/mRBS-TetR	sRBS	Red	PstI and SpeI
sRBS/mRBS-TetR	mRBS	Red	PstI and SpeI
sRBS/mRBS-TetR	TetR	Tango	XbaI and PstI
TetR-dT	TetR	Red	EcoRI and SpeI
TetR-dT	dT	Orange	XbaI and EcoRI
dT-pTet	dT	Red	EcoRI and SpeI
dT-pTet	pTet	Orange	XbaI and EcoRI
pLAcI-sRBS/mRBS	pLacI	Red	EcoRI and SpeI
pLAcI-sRBS/mRBS	sRBS	Orange	XbaI and EcoRI
pLAcI-sRBS/mRBS	mRBS	Orange	XbaI and EcoRI
Mms6-PET28(a)	PET28(a)	Orange	NotI

• For all reactions
  • 158 (µL) Milli-Q H₂O
  • 10 (µL) Buffer
  • 0.5(µL) of each enzyme
  • 10 (µL) pDNA

Restriction was incubated for 1 hour at 37^oC


Objective: Run PCR of pBAD, TetR, dT, pLacI, and mms6.
Method: Used PCR thermocycler iGEM program 7

Component	Volume per tube (µL)	Master Mix (x6)
MilliQ H2O	41.8	250.8
10x Pfu Buffer with MgSO4	5	30
dNTPs	1	6
Forward Primer	0.5	3
Reverse Primer	0.5	3
Template DNA	1	-
Pfu DNA polymerase	0.2	-
Total	50	292.8

• Added 48.8 µL of Master Mix to each PCR reaction
  
  
  

Objective: Complete maxipreps of Lumazine (K249002), EYFP (E0030), and ECFP (E0020) and run on 1% agarose gel.
Method:

Lane	Sample	Components (µL)
1	1 kb ladder	2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H2O
2	ECFP	2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
3	EYFP	2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
4	Lumazine	2 DNA + 2 loading dye (5x) + 2 MilliQ H2O

• Ran at 100V for 42 minutes. Stained in EtBr for 15 minutes.
  

Results: AGAROSE GEL PICTURE


Objective: Ligate dT into pSB1C3.
Method:

• PCR amplify and purify both pSB1C3 and dT
• Restrict both with EcoRI and PstI
• Restrict pSB1C3 with DpnI
• Ligate pSB1C3 and dT
  

Restriction:

Restriction	MilliQ H2O (µL)	Buffer Orange (µL)	pDNA (µL)	Enzyme (µL)
pSB1C3	79.25	10	10	0.25 EcoRI + 0.25 PstI + 0.25 DpnI
pSB1C3 control	80	10	10	-
dT	79.50	10	10	0.25 EcoRI + 0.25 PstI
dT control	80	10	10	-

• Restriction were incubated at 37^oC for 90 minutes.
• Enzymes were heat killed for 20 minutes at 80^oC.

August 3, 2010 Evening

(in lab: KG, JS)

Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.

Lane	Contents	Result
1	1kb ladder
2	pTet (XbaI, EcoRI)	good
3	pTet (orange buffer)	---
4	dT (XbaI, EcoR)	no good
5	dT (orange buffer)	---
6	mRBS (SpeI, PstI)	good
7	mRBS (red buffer)	---
8	sRBS (SpeI, PstI)	good
9	sRBS (red buffer)	---
10	mRBS (XbaI, EcoRI)	good
11	mRBS (orange buffer)	---
12	sRBS (XbaI, EcoRl)	good
13	sRBS (orange buffer)	---
14	pet28(a)	good
15	100 bp ladder
16	PSB1C3	not good
17	PSB1C3 restriction digest	---

Lane	Contents	Result
1	TetR (EcorI, SpeI)	good
2	TetR (red buffer)	---
3	pLacI (EcoRI, SpeI)	good
4	pLacI (red buffer)	---
5	Mms6	can not tell
6	Mms6 control	---
7	TetR (Xbal, PstI)	good
8	TetR (tango buffer)	---
9	pBAD (EcoRI, SpeI)	good
10	pBAD (red buffer)	---
11	dT (EcoRI, SpeI)	good
12	dT (red buffer)	---
13	pLacI (2)	?
14	dT control	not good
15	dT restriction
16	100 bp ladder
17	dT PCR product	good
18	Mms6 PCR product	good
19	pBAD PCR product	good
20	pLacI PCR product	good

---

Objective: To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3

Method: Ligation of Plasmid DNA

15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)

August 4, 2010

(in lab: JV)

Objective: PCR analysis of ligation product of August 3, 2010

• Ligations
  • pBAD-mRBS
  • pBAD-SRBS
  • SRBS-tetR
  • mRBS-TetR
  • dt-pTet
  • mms6-pET-28a
  • dt-pSBIC3
  • pLacI-SRBS
• Controls
  • pBAD
  • TetR
  • TetR
  • pLacI
  • mms6

Method:
PCR: Thermocycler set to iGEM program 7

Component	1X(µL)	Master Mix(x16)(µL)
Milli-Q H2O	41.8	668.6
10x Pfu Buffer with MgSO4	5	80
dNTPs	1	16
Forward Primer	0.5	8
Reverse Primers	0.5	8
Template DNA	1	16
Pfu polymerase	0.2	3.2

2.5% agarose gel(1x TAE)

lane	contents	Successful Ligation ?
1	50bp ladder	---
2	dt pSBIC3	---
3	dt pTet	x
4	dt control	---
5	sRBS-TetR	x
6	mRBS-TetR	?
7	TetR control	---
8	pLacI-mRBS	x
9	pLacI-sRBS	?
10	pLacI control	---
11	Mms6 pET-28a	no band
12	Mms6 control	---
13	pBad-SRBS	x
14	pBad-mRBS	x
15	pBad control	---

• Ran at 100V for 70 minutes.

Results: AGAROSE GEL PICTURE


Objective: Transform the successful ligations

Method: used Competent Cell Transformation protocol

• changes:
  • used 50µL aliquottes of DH5&alpha
  • did not pipette up and down once, the cells were just swirled 3 times
  • added 400µL SOC media, shoock at 37⁰C for 90 min
  • platted 250µL and 150µL
    

Incubated from 12:00AM to 4;00 PM

results
contents	&250µL	150µL
dt-pTet	good	x
- control	x	x
mms6-pET-28a	good	good
dt-pSBIC3	x	x
mRBS-TetR	good	good
pLacI-mRBS	good	good
SRBS-TetR	x	x
pBAD-SRBS	good	good
+ contol	good	good

August 6/2010 Evening

Objective: Attempt colony pcr for rapid screening

Method: Followed two protocols from openwet

• Knight Protocol
  
  • place 20(µL) sterile H₂O in 0.6mL sterile tube
  • with P10 pipette set to 3(µL) dip tip into colony
  • place pipette tip into water and pipette up and down 20 times(this can be stored at 4⁰C for inoculation of overnight 5mL cultures)
    

• Endy Protocol
  • place 50(µL) sterile H₂O in 0.6mL sterile tube
  • with PLO pipette (set 3(µL)) dip sterile tip into colony
  • place pipette tip into water and pipette up and down 20 times

Knight cont'd	Endy cont'd
setup 20(µL) reaction	setup 20(µL) reaction
1(µL) colony suspension	2(µL) colony suspension
2(µL) 10x p.fu (+Mg SO4	2(µL) 10x p.fu (+Mg SO4
2(µL) dNTP	2(µL) dNTP
1.25(µL)VF2 Primer (10(µM)	0.75(µL)VF2 Primer (10(µM)
1.25(µL)VF Primer (10(µM)	0.75(µL)VF Primer (10(µM)
0.2(µL) Pfu polymerase	0.2(µL) Pfu polymerase
11.8 Milli-Q H2O	12.6 Milli-Q H2O

• as control for each rxn used equal volume of mRBS maxiprep

Knight cont'd	Endy cont'd
cycling conditions	cycling conditions
950C for 15 minutes	950C for 6 minutes
*940C for 30 seconds	**950C for 30 seconds
*560C for 30 seconds	**560C for 30 seconds
*680C for 1 minutes	**700C for 1 minutes
680C for 20 minutes	700C for 10 minutes

(*) were run 39 times
(**) were run 35 times

Made the following program (called COLONYY) Lid preheat 98⁰C

• 98⁰C for 15 minutes
• 98⁰C for 30 seconds
• 56⁰C for 30 seconds
• 68-70⁰C gradient for 1 minute
• 68-70⁰C gradient for 20 minute
• 4⁰C indefinte

bold selections were cycled 39 times

Objective: Analyzed PCR products on 2.5% TAE Agarose gel.

lane	sample	loaded
1	50bp ladder	1(µL) ladder, 1(µL) dye, 4(µL) H20
2	Knight control	5(µL) sample, 1(µL)dye
3	Knight colony	5(µL) sample, 1(µL)dye
4	Endy control	5(µL) sample, 1(µL)dye
5	Endy colony	5(µL) sample, 1(µL)dye
6	50bp ladder	1(µL) ladder, 1(µL) dye, 4(µL) H20
7	lumazine(justin's)	5(µL) sample, 1(µL)dye

Repeat gel with template controls

lane	sample	loaded
1	50bp ladder	0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20
2	Endy Template	5(µL) colony suspension, 1(µL)dye, 4(µL) H20
3	Endy mRBS Control (PLR)	5(µL) sample, 1(µL)dye
4	Endy mRBS-TetR colony(PCR)	5(µL) sample, 1(µL)dye
4	mRBS template	0.5(µL) sample, 1(µL)dye, 4(µL) H20
5	Knight Template	0.25(µL) colony suspension, 1(µL)dye, 4(µL) H20
6	Knight mRBS control	5(µL) ladder, 1(µL) dye
7	Knight-mRBS-TetR colony	5(µL) sample, 1(µL)dye
1	1Kb ladder	0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20

• ran at 100V for 75 minutes

Aug 9/2010

(In Lab: AV)

Objective: Prepared glycerol stocks & Miniprepped the following using the Qiagen Protocol.

pLacI-mRBS Colony 1

pLacI-mRBS Colony 2

pLacI-sRBS Colony 2

pLacI-sRBS Colony 3

pBAD-mRBS Colony 1

pBAD-mRBS Colony 2

pBAD-sRBS Colony 1

pBAD-sRBS Colony 2

dT-pTet Colony 1

dT-pTet Colony 3

mRBS-TetR Colony 1

mRBS-TetR Colony 3

---

Objective: To determine which of the previous ligations worked.

Method: Restricted with single cutter and double cutter.

Restriction Reaction (SINGLE)

Ingredient	Volume(µL)
MilliQ H20 Water	15.75
Orange Buffer (10x)	2
pDNA	2
EcoRI	0.25

Restriction Reaction (DOUBLE)

Ingredient	Volume(µL)
MilliQ H20 Water	15.50
Orange Buffer (10x)	2
pDNA	2
EcoRI	0.25
PstI	0.25

Unrestricted Control

Ingredient	Volume(µL)
MilliQ H20 Water	16
Orange Buffer (10x)	2
pDNA	2

DNA was restricted for 1 hour at 37^oC.

Analyzed results on a 2% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA. Load order as follows:

Lane	Contents
1	1kb ladder
2	50 bp ladder
3	dT-pTet 1 DRD
4	dT-pTet 1 SRD
5	dT-pTet 1 URD
6	dT-pTet 3 DRD
7	dT-pTet 3 SRD
8	dT-pTet 3 URD
9	pLacI-mRBS 1 DRD
10	pLacI-mRBS 1 SRD
11	pLacI-mRBS 1 URD
12	pLacI-mRBS 2 DRD
13	pLacI-mRBS 2 SRD
14	pLacI-mRBS 2 URD
15	pLacI-sRBS 1 DRD
16	pLacI-sRBS 1 SRD
17	pLacI-sRBS 1 URD
18	pLacI-sRBS 3 DRD
19	pLacI-sRBS 3 SRD
20	pLacI-sRBS 3 URD

Lane	Contents
1	1 kb ladder
2	50 bp ladder
3	pBAD-mRBS 1 DRD
4	pBAD-mRBS 1 SRD
5	pBAD-mRBS 1 URD
6	pBAD-mRBS 2 DRD
7	pBAD-mRBS 2 SRD
8	pBAD-mRBS 2 URD
9	pBAD-sRBS 1 DRD
10	pBAD-sRBS 1 SRD
11	pBAD-sRBS 1 URD
12	pBAD-sRBS 2DRD
13	pBAD-sRBS 2 SRD
14	pBAD-sRBS 2 URD
15	mRBS-TetR 1 DRD
16	mRBS-TetR 1 SRD
17	mRBS-TetR 1 URD
18	mRBS-TetR 3 DRD
19	mRBS-TetR 3 SRD
20	mRBS-TetR 3 URD

GEL PICTURE!

---

Aug 9/2010 Evening

(In Lab: JV, AS)

Objective: To ligate: lumazine into vector upstream of dT. Lumazine and mms6 into pET28a.

Method:

• Restrictions
  
  • Restrict Lumazine wit EcoRI and SpeI (Red Buffer)
    
  • Restrict the dT with XbaI and EcoRI (Orange Buffer)
    
  • Restrict Lumazine Synthase with NotI (Red Buffer)
    
  Set up reactions as follows:
  

  Component	Volume (µL)
  MilliQ H2O	15.6 or 15.8
  Buffer	2
  pDNA	2
  Enzyme	0.20 + 0.20

  Incubated reactions for 60 minutes at 37^oC
  

• Ligation
  Reaction set up as follows:
  • T4 DNA ligase - 0.25µL
    
  • DNA 1 - 8µL
    
  • DNA 2 - 8µL
    
  • 10x Ligation Buffer - 2µL
    
  • MilliQ H₂O - 1.75µL
    
  Incubated reactions overnight at room temperature.

  Aug 10/2010

  (In Lab: JV)
  

  Objective: Reran large gel from Aug 9/2010.
  

  Load order was as follows:
  

  Lane	Contents
  1	50 bp ladder
  2	pBAD-mRBS 2 URD
  3	pBAD-mRBS 2 SRD
  4	pBAD-mRBS 2 DRD
  5	pLacI-sRBS 3 URD
  6	pLacI-sRBS 3 SRD
  7	pLacI-sRBS 3 DRD
  8	pLacI-mRBS 2 URD
  9	pLacI-mRBS 2 SRD
  10	pLacI-mRBS 1 DRD
  11	dT-pTet 3 URD
  12	dT-pTet 3 SRD
  13	dT-pTet 3 DRD
  14	pBAD-mRBS 1 URD
  15	pBAD-mRBS 1 SRD
  16	pBAD-mRBS 1 DRD
  17	pLacI-sRBS 2 URD
  18	pLacI-sRBS 2 SRD
  19	pLacI-sRBS 2 DRD
  20	1 kb ladder

  
  

  Lane	Contents
  1	50 bp ladder
  2	pLacI-mRBS 1 URD
  3	pLacI-mRBS 1 SRD
  4	pLacI-mRBS 1 DRD
  5	dT-pTet 1 URD
  6	dT-pTet 1 SRD
  7	dT-pTet 1 DRD
  8	mRBS-TetR 3 URD
  9	mRBS-TetR 3 SRD
  10	mRBS-TetR 3 DRD
  11	mRBS-TetR 1 URD
  12	mRBS-TetR 1 SRD
  13	mRBS-TetR 1 DRD
  14	pBAD-sRBS 2 URD
  15	pBAD-sRBS 2 SRD
  16	pBAD-sRBS 2 DRD
  17	pBAD-sRBS 1 URD
  18	pBAD-sRBS 1 SRD
  19	pBAD-sRBS 1 DRD
  20	1 kb ladder

  
  

  GEL PICTURE!

  Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/August"