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From 2010.igem.org

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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)

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 37⁰C

Harland to read over and make sure what i wrote is correct

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

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.