Team:Newcastle/31 August 2010

From 2010.igem.org

(Difference between revisions)
(Aim)
(Glycerol stocks)
 
(51 intermediate revisions not shown)
Line 1: Line 1:
{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
-
=Amplification of the plasmid PSB1C3 for ''rocF'' BioBrick=
+
=Glycerol stocks=
-
 
+
Today we made [[Team:Newcastle/Glycerol_stocks|glycerol stocks]] of our filamentous ''Bacillus subtilis'' 168 strain.
-
==Aim==
+
-
The aim of this experiment is to amplify plasmid linearized pSB1C3 for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of a single Phusion PCR using old primers.
+
-
 
+
-
==Materials and Protocol==
+
-
Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:
+
-
 
+
-
===PCR===
+
-
{|border=1
+
-
|-
+
-
!'''Tube'''
+
-
!'''Part to be amplified'''
+
-
!'''DNA fragment consisting the part'''
+
-
!'''Forward primer'''
+
-
!'''Reverse Primer'''
+
-
!'''Melting Temperature (Tm in °C) '''
+
-
!'''Size of the fragment (in bp)'''
+
-
!'''Extension time* (in seconds)'''
+
-
|-
+
-
|1
+
-
|Plasmid Vector
+
-
|pSB1C3
+
-
|P1V1 forward
+
-
|P2V1 reverse
+
-
|58
+
-
|2072 approx.
+
-
|60
+
-
|-
+
-
|2
+
-
|Pspacoid Promoter
+
-
|pMutin4
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|49
+
-
|106 approx.
+
-
|15
+
-
|-
+
-
|3
+
-
|1st fragment of ''rocF'' CDS
+
-
|''B. subtilis'' 168 chromosome
+
-
|P1S1 forward
+
-
|P2S1 reverse
+
-
|58
+
-
|246 approx.
+
-
|15
+
-
|-
+
-
|4
+
-
|2nd fragment of ''rocF'' CDS
+
-
|''B. subtilis'' 168 chromosome
+
-
|P3S2 forward
+
-
|P4S2 reverse
+
-
|65
+
-
|597 approx.
+
-
|20
+
-
|-
+
-
|5
+
-
|3rd fragment of ''rocF'' CDS
+
-
|''B. subtilis'' 168 chromosome
+
-
|P5S3 forward
+
-
|P6S3 reverse
+
-
|66
+
-
|125 approx.
+
-
|15
+
-
|-
+
-
|6
+
-
|Double Terminator
+
-
|pSB1AK3 consisting BBa_B0014 biobrick
+
-
|P1T1 forward
+
-
|P2T1 reverse
+
-
|56
+
-
|116 approx.
+
-
|15
+
-
|}
+
-
 
+
-
'''Table 1''': Table represents 6 different Phusion PCR reactions and the parts which are amplified so that they can be ligated together with the help of Gibson Cloning method.
+
-
* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
+
-
* For learning about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
+
-
 
+
-
==Discussion==
+
-
All the 6 Phusion PCR reactions were done however, gel electrophoresis was not done today to check whether the fragments have actually amplified or not.
+
-
 
+
-
==Conclusion==
+
-
Tomorrow 5th August, 2010, we would be running gel electrophoresis to check the outcome of the 6 PCR reactions and later all the fragments will be ligated with help of Gibson protocol.
+
-
 
+
-
 
+
-
 
+
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 01:20, 28 October 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

Glycerol stocks

Today we made glycerol stocks of our filamentous Bacillus subtilis 168 strain.

Newcastle University logo.png    Newcastle cbcb logo.pngNewcastle Biomedicine logo.gif    Team Newcastle CEG logo.gif
Newcastle iww logo.jpg  UNIPV Pavia Logo.gif  Newcastle BBSRC.gif    Newcastle Genevision logo.png Newcastle WelcomeTrust.jpg
FaceBook Icon