Team:Newcastle/9 August 2010

From 2010.igem.org

(Difference between revisions)
(→Conclusion)
 
(39 intermediate revisions not shown)
Line 1: Line 1:
{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
-
=Transformation of pVeg and lacI=  
+
=Subtilin immunity part=
-
==Results/Aims==
+
Today we were working on our subtilin immunity part cloning strategy.
-
 
+
-
* There are colonies on all 3 plates for pVeg.
+
-
* For ''lacI'', there is only one colony on one of the normal plates and more on the concentrated one.
+
-
 
+
-
==Discussion==
+
-
 
+
-
* For pVeg, we will set up an [[Team:Newcastle/Growing_an_overnight_cultures|overnight broth culture]] from the colonies for [[Team:Newcastle/Qiagen_Minipreps|plasmid extraction]]
+
-
* To show that we have sucessfully transformed from ''lacI'', we will select 4 colonies and plate them on a section of an agar plate as well as setting up overnight broth cultures.
+
-
 
+
-
==Conclusion==
+
-
 
+
-
Please refer to [[Team:Newcastle/10_August_2010|10.08.10]].
+
-
 
+
-
=Transformation of lacI=
+
-
 
+
-
==Aims==
+
-
 
+
-
To transform some competent ''E.coli'' DH5α with the ligation products: 1:3, 1:5 and the vector from the ligation on [[Team:Newcastle/6_August_2010|06.08.10]].
+
-
 
+
-
==Materials and Protocol==
+
-
 
+
-
Please refer to [[TeamNewcastleTransformation_of_E._coli|Transformation of E.coli]].
+
-
 
+
-
==Result==
+
-
 
+
-
Please refer to [[Team:Newcastle/10_August_2010|10.08.10]] for result.
+
-
 
+
-
=Amplification of Pspac_oid promoter by PCR=
+
-
 
+
-
==Aim==
+
-
The aim of this experiment is to amplify Pspac_oid promoter fragment from plasmid pMutin4 for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of 2 different Phusion PCR.
+
-
 
+
-
==Materials and Protocol==
+
-
Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 4 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
+
-
|Pspacoid Promoter
+
-
|pMutin4
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|58
+
-
|106 approx.
+
-
|15
+
-
|-
+
-
|2
+
-
|Pspacoid Promoter
+
-
|pMutin4
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|59
+
-
|106 approx.
+
-
|15
+
-
|}
+
-
 
+
-
'''Table 1''': Table represents 4 different Phusion PCR reactions for the amplification of Pspac_oid promoter, so that it can be ligated together with other fragments for the construction of ''rocF'' 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 2 Phusion PCR reactions were done however, gel electrophoresis will be done later today, to check whether the fragments have actually amplified or not.
+
-
 
+
-
==Conclusion==
+
-
Today afternoon, we would be running gel electrophoresis to check the outcome of the 2 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
+
-
 
+
-
 
+
-
=Amplification of Pspac_oid promoter by PCR=
+
-
 
+
-
==Aim==
+
-
The aim of this experiment is to amplify Pspac_oid promoter fragment from plasmid pMK-RQ containing Biobrick ''kinA'' and plasmid pMK-RQ containing stochastic switch developed by Team Newcastle 2009 for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of 4 different Phusion PCR.
+
-
 
+
-
==Materials and Protocol==
+
-
Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 2 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
+
-
|Pspacoid Promoter
+
-
|Plasmid containing ''kinA''
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|58
+
-
|106 approx.
+
-
|15
+
-
|-
+
-
|2
+
-
|Pspacoid Promoter
+
-
|Plasmid containing ''kinA''
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|59
+
-
|106 approx.
+
-
|15
+
-
|-
+
-
|3
+
-
|Pspacoid Promoter
+
-
|Plasmid containing stochastic switch
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|58
+
-
|106 approx.
+
-
|15
+
-
|-
+
-
|1
+
-
|Pspacoid Promoter
+
-
|Plasmid containing stochastic switch
+
-
|P1P1 forward
+
-
|P2P1 reverse
+
-
|59
+
-
|106 approx.
+
-
|15
+
-
|}
+
-
 
+
-
'''Table 2''': Table represents 2 different Phusion PCR reactions for the amplification of Pspac_oid promoter, so that it can be ligated together with other fragments for the construction of ''rocF'' 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 4 Phusion PCR reactions were done however, gel electrophoresis will be done later today, to check whether the fragments have actually amplified or not.
+
-
 
+
-
==Conclusion==
+
-
Today afternoon, we would be running gel electrophoresis to check the outcome of the 4 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
+
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 23:03, 27 October 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

Subtilin immunity part

Today we were working on our subtilin immunity part cloning strategy.

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
Retrieved from "http://2010.igem.org/Team:Newcastle/9_August_2010"