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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

AV

Objective: To do Glycerol stocks/miniprep the following

@@ Line 98: / Line 98: @@
 Back to [[Team:Lethbridge/Notebook/Lab_Work|Lab Work]]
-=August=
+=<font color="white">August=
-==August 3, 2010==
+==<font color="white">August 3, 2010==
 (in Lab: HB, AV, JV)<br>
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 *Enzymes were heat killed for 20 minutes at 80<sup>o</sup>C.
-==August 3, 2010 Evening==
+==<font color="white">August 3, 2010 Evening==
 (in lab: KG, JS)<br>
@@ Line 236: / Line 236: @@
 (&micro;L) pDNA in plasmid, and 15 (&micro;L) of pDNA biobrick)<br>
-==August 4, 2010==
+==<font color="white">August 4, 2010==
 (in lab: JV)<br>
@@ Line 317: / Line 317: @@
-==August 6/2010 Evening==
+==<font color="white">August 6/2010 Evening==
 <b>Objective:</b> Attempt colony pcr for rapid screening<br>
@@ Line 401: / Line 401: @@
 *ran at 100V for 75 minutes
-==Aug 9/2010==
+==<font color="white">Aug 9/2010==
 AV
 <center><b> Objective:</b> To do Glycerol stocks/miniprep the following</center>

Team:Lethbridge/Notebook/Lab Work/August