"

From 2014.igem.org

Revision as of 15:33, 9 September 2014 (view source) MelaCriq (Talk | contribs) (→cloning) ← Older edit		Revision as of 15:44, 9 September 2014 (view source) MelaCriq (Talk | contribs) (→cloning) Newer edit →
Line 32:		Line 32:
	====cloning====		====cloning====
-	Cloning of PCR purification in a TA plasmid	+	Cloning of PCR purification in a TA plasmid (Topo plasmid)
	First I had to add AAA to the PCR		First I had to add AAA to the PCR

---

Revision as of 15:44, 9 September 2014

Contents 1 Lab Work 1.1 D- Lemon Scent 1.1.1 Verfication of the pPS3/4/5 plasmid 1.1.2 PCR verification 1.1.3 cloning 1.1.4 Transformation

Lab Work

D- Lemon Scent

By Mélanie

Verfication of the pPS3/4/5 plasmid

Due to the strange results obtained last day, I do a PCR of the insert with the differents plasmid : I use the same PCR condition than September 2 but with the right primer and plasmid

We can see that we don't have any insert in our plasmid

PCR verification

We find some other PCR of BBa K762100 and GS and BBa_K517003 so we check it by electrophoresis

And I purify the PCR

Well 1 = Ladder Well 2 = GS Well 3 = PS Well 4 = CAD

We can see that we don't have lost a lot of our PCR product

cloning

Cloning of PCR purification in a TA plasmid (Topo plasmid) First I had to add AAA to the PCR

component	volume
buffer	1μl
dATP	1μl
PCR	7.5μl
Taq	0.5μl

72° 15'

and

component	volume
H2O	1μl
buffer	1μl
vector	1μl
cloning	1μl

30' at room temperature

Transformation

Transformation with competent bacteria

add DNA (Top vector our pPSII)

30' on ice 45' 42°c 30' at 37°

spread on petri dish