Team:Paris/August 4

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Latest revision as of 15:22, 6 August 2008

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PCR Screening of Ligation Transformants

Use of 8 clones of Ligation transformants n°L100 for screening PCR

Protocol of screening PCR

Mix

Name	Vol (µl)	Concentration
Quick Load	25µl	2X
OligoF_VF2 (O18)	1µl	10µM
OligoR_VR (O19)	1µl	10µM
water	23µl

50µl of Mix PCR by tube/clone
one toothpick of each clone's colony by tube
Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Conditions of electrophoresis

10µl of ladder 1 kb
20µl of screening PCR
migration ~30min at 100W on 0,8% gel

Results for L100

L100= D110 + D130 = RBS-tetR-ECFP-Ter

Band	1-->8
Expected size	1042 pb
Measured size	1100 pb
Gel : L100(1-8)

==> Conclusion : we obtain a low quantity of PCR products, but at the good size.
So now, we know that we can amplify the clones that we want for L100 transformants bacteria.

@@ Line 49: / Line 49: @@
 ===Results for L100===
-L100= D110 + D130 = '''RBS-tetR-ECFP-Ter''' [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
+L100= D110 + D130 = '''RBS-tetR-ECFP-Ter''' [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 {| border="1"
@@ Line 63: / Line 63: @@
 |colspan="8"|[[Image:KR000101_1.jpg|thumb|'''Gel : L100(1-8)''']]
 |}
+==> '''Conclusion :''' we obtain a low quantity of PCR products, but at the good size.
+<br>So now, we know that we can amplify the clones that we want for L100 transformants bacteria.