From 2014.igem.org

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19^th June

• PCR of our inserts 40 and 60


• Digestion of psB1C3 and our inserts 20-40-60
  
  Concentration PCR 40 = 321,3ng/µl
  
  PCR 60 = 521,0ng/µl
  
  Concentration psB1C3 = 96,5ng/µl -> plate 4 kit 2014
  
  Digestion psB1C3 :



H2O (qsp 20µl)	7,5µl
Buffer 2	2µl
XbaI	0,25µl
SpeI	0,25µl
Vector	10µL

With vector = 5,18µL psB1C3 + 4,82µL H2O


• Ligation
We need to have a ratio [insert] = 3*[plasmid]
We use the website promega

• psB1C3 = 2270pb
• VPGXG 20 = 436pb
• VPGXG 40 = 736pb
• VPGXG 60 = 1036pb


So [plasmid] = 0,96ng/µl (via nanodrop)

= 0,017 pmol/µl (via the website)
= 0,034 pmol/ 2µl

So we need : 0,017*3=0,051pmol/µl  0,017 * 6 = 0,102pmol/2µl

	20	40	60
Digestion	2µl	2µl	2µl
Insert	1,13µl	1,92µl	2,75µl
Buffer ligase 10X	1µl	1µl	1µl
T4 ligase	0,5µl	0,5µl	0,5µl
H2O qsp 10µl

• SDS-PAGE Tris-Glycine

• Design of oligo with DNA works (website)