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Contents 1 August 1.1 August 3, 2010 1.2 August 3/2010 Evening 1.3 August 4/2010 1.4 August 4/2010

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:K.G J.S)

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 aug 3/2010

will PCR:

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

• controls
  • pBAD
  • TetR
  • TetR
  • pLacI
  • Mms6

	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

Objective: Run PCR samples on a 2.5% agarose gel(1x TAE)for 70 minutes

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	---

all ligations were transformed

Objective: Transform

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	:)	x
- control	x	x
mms6-pET-28a	:)	:)
dt-pSBIC3	x	x
mRBS-TetR	:)	:)
pLacI-mRBS	:)	:)
SRBS-TetR	x	x
pBAD-SRBS	:)	:)
+ contol	:)	:)

"AUG 4" is not yet done

August 4/2010

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.