Team:Groningen/1 June 2010

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We've started working on the wiki, been preparing plasmids and are settling in our ''office space''. Note to self: should have brought coffee machine
We've started working on the wiki, been preparing plasmids and are settling in our ''office space''. Note to self: should have brought coffee machine
-
Week 22 [br]
 
-
Week 23
+
'''Week 22, Arend Jan'''
 +
The biobrick prefix and suffix are introduced into the pNZ8901 expression vector behind the spaS promoter using PCR and primers with 5’ overhangs containing the prefix and suffix. In this way, any biobrick with a RBS followed by a coding sequence can be brought to expression upon subtilin induction.
 +
For optimization taq polymerase (fermentas) is used first.
 +
PCR:
 +
<pre>Component         µl Final concentration
 +
Primer pNZ89bbs-for1 5 300nM
 +
Primer pNZ89bbs-rev1 5 300nM
 +
10x Taq buffer(-MgCl2) 5 1x
 +
dNTP’s                 2 200µM
 +
MgCl2                 3 1.5mM
 +
Template         0.5 ~14ng
 +
Taq                 0.5 2.5u
 +
MQ                 29
 +
- 94°C, 3’
 +
- 94°C,  30’’
 +
30X - 50°C,  30’’
 +
- 72°C,  1’30’’
 +
- 72°C,  10’
 +
</pre>
 +
No product. Likely the elongation step was too short for taq.
 +
Repeat of previous PCR using the following cycling conditions.
 +
<pre>- 94°C, 3’
 +
- 94°C,  30’’
 +
30X - 50°C,  30’’
 +
- 72°C,  3’
 +
- 72°C,  10’</pre>
 +
 +
[[Image:02-06-10gn.jpg|200px|thumb|left|Please excuse the image quality, will get better]]
 +
 +
<br style="clear: both" />
 +
Product is ~4kb instead of 3,2. This has been observed before and plasmid is ok to use.
 +
Repeated PCR with pfu polymerase (fermentas) to prevent point mutations in plasmid.
 +
 +
 +
PCR:
 +
<pre>Component         µl Final concentration
 +
Primer pNZ89bbs-for1 5 300nM
 +
Primer pNZ89bbs-rev1 5 300nM
 +
10x pfu buffer(-MgSO4) 5 1x
 +
dNTP’s                 2 200µM
 +
MgCl2                 3 1.5mM
 +
Template        0.5 ~14ng
 +
Pfu polymerase    1 2.5u
 +
MQ                 28.5
 +
 +
- 94°C, 3’
 +
- 94°C,  30’’
 +
30X - 50°C,  30’’
 +
- 72°C,  1’30’’
 +
- 72°C,  10’ </pre>
 +
 +
 +
[[Image:03-06-10gn.jpg|200px|thumb|left|]]
 +
 +
<br style="clear: both" />
 +
 +
Good bands. The 5’of the primers were designed with BamHI sites for easy circularization of the plasmid. The PCR product was cleaned (High Pure PCR Product Purification kit, Roche) and digested with BamHI.
 +
 +
 +
<pre>
 +
- 22ul plasmid(~500ng)
 +
- 2.5ul buffer G
 +
- 0.5ul BamHI (fermentas)
 +
</pre>
 +
 +
 +
Digestion was cleaned (Roche kit) and a ligation was performed for circularization
 +
 +
 +
<pre>
 +
- 10ul plasmid(~40ng)
 +
- 5ul 10x T4 ligase buffer
 +
- 5ul T4 ligase (fermentas, LC)
 +
- 30ul MQ
 +
</pre>
 +
 +
 +
20ul was used for a transformation of an E.coli top10 strain made competent using the CaCl2 method. Only a few colonies grew on the positive control transformation. Should be repeated with good competent cells.

Latest revision as of 01:12, 8 October 2010

iGEM Groningen 2010

Hydrophobofilm
pushing coatings into a greener future

We've started working on the wiki, been preparing plasmids and are settling in our office space. Note to self: should have brought coffee machine


Week 22, Arend Jan


The biobrick prefix and suffix are introduced into the pNZ8901 expression vector behind the spaS promoter using PCR and primers with 5’ overhangs containing the prefix and suffix. In this way, any biobrick with a RBS followed by a coding sequence can be brought to expression upon subtilin induction.


For optimization taq polymerase (fermentas) is used first.

PCR:

Component	        µl	Final concentration
Primer pNZ89bbs-for1	5	300nM
Primer pNZ89bbs-rev1	5	300nM
10x Taq buffer(-MgCl2)	5	1x
dNTP’s	                2	200µM
MgCl2	                3	1.5mM
Template	        0.5	~14ng
Taq	                0.5	2.5u
MQ	                29	

- 94°C,	 3’
	- 94°C,  30’’
30X	- 50°C,  30’’
	- 72°C,  1’30’’
- 72°C,  10’


No product. Likely the elongation step was too short for taq.

Repeat of previous PCR using the following cycling conditions.


- 94°C,	 3’
	- 94°C,  30’’
30X	- 50°C,  30’’
	- 72°C,  3’
- 72°C,  10’


Please excuse the image quality, will get better


Product is ~4kb instead of 3,2. This has been observed before and plasmid is ok to use. Repeated PCR with pfu polymerase (fermentas) to prevent point mutations in plasmid.


PCR:

Component	        µl	Final concentration
Primer pNZ89bbs-for1	5	300nM
Primer pNZ89bbs-rev1	5	300nM
10x pfu buffer(-MgSO4)	5	1x
dNTP’s	                2	200µM
MgCl2	                3	1.5mM
Template         	0.5	~14ng
Pfu polymerase    	1	2.5u
MQ	                28.5	

- 94°C,	 3’
	- 94°C,  30’’
30X	- 50°C,  30’’
	- 72°C,  1’30’’
- 72°C,  10’ 


03-06-10gn.jpg


Good bands. The 5’of the primers were designed with BamHI sites for easy circularization of the plasmid. The PCR product was cleaned (High Pure PCR Product Purification kit, Roche) and digested with BamHI.


- 22ul plasmid(~500ng) 
- 2.5ul buffer G
- 0.5ul BamHI (fermentas)


Digestion was cleaned (Roche kit) and a ligation was performed for circularization


- 10ul plasmid(~40ng)
- 5ul 10x T4 ligase buffer
- 5ul T4 ligase (fermentas, LC)
- 30ul MQ


20ul was used for a transformation of an E.coli top10 strain made competent using the CaCl2 method. Only a few colonies grew on the positive control transformation. Should be repeated with good competent cells.





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