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Revision as of 01:33, 25 August 2010 (view source) Liszabruder (Talk | contribs) ← Older edit		Revision as of 02:06, 25 August 2010 (view source) Katherine.gzyl (Talk | contribs) (→August 3/2010) Newer edit →
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	* For all reactions		* For all reactions
-	** 158 (&micro;L) MilliQ H2O	+	** 158 (&micro;L) Milli-Q H<sub>2</sub>O
	** 10 (&micro;L) Buffer		** 10 (&micro;L) Buffer
	** 0.5(&micro;L) of each enzyme		** 0.5(&micro;L) of each enzyme
	** 10 (&micro;L) pDNA		** 10 (&micro;L) pDNA
-	Restriction was incubated for 1 hour at 37 0C	+	Restriction was incubated for 1 hour at 37<sup>0</sup>C
		+
		+
		+	<font color ="red">Harland to read over and make sure what i wrote is correct</font><br>
		+
		+	==August 3/2010 Evening==
		+	(In Lab:K.G J.S)<br>
		+
		+	<b>Objective:</b> To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet<br>
		+
		+	<b>Method:</b> Used a 1.5% agarose gel with 2 (&micro;L) of loading dye and 10 (&micro;L) of pDNA.<br>

---

Revision as of 02:06, 25 August 2010

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.