Team:Lethbridge/Notebook/Lab Work/August

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Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

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

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.

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

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

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

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

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	-

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	---

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

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	---

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

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

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

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

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

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