''roc''F miniprep: we aim to purify ''roc''F in p.....
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=Checking for transformants=
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===Materials and protocol===
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Today we checked for transformants from the filamentous ''Bacillus subtilis'' transformation (with pMutin4) we performed yesterday.
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[[Team:Newcastle/Qiagen_Minipreps#Plasmid_extraction|See Mini prep protocol]]
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===Results===
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====Nanodrop====
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''roc''F
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Concentration of DNA in ng/µl
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#93.1
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#102.0
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#99.4
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#101.0
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#98.8
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#104.7
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#93.1
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#57.9
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#81.9
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=Subtilin Immunity BioBrick=
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=== Aims ===
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Overnight cultures of all the colonies from the 'SI 1/9/10' plates was carried out today.
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=== Methods ===
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The [[Team:Newcastle/Growing_an_overnight_cultures| Growing Overnight Cultures]] protocol was followed. The colonies were grown on LB with chloramphenicol. Altogether 16 colonies were produced on the plates and were left overnight at 37°C.
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=== Results, Discussion and Conclusion ===
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After the overnight cultures have been left overnight, minipreps of the 16 colonies will be performed [[Team:Newcastle/7 September 2010#Subtilin Immunity BioBrick| tomorrow]].
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=Sucrose/Levan's glue=
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The sucrose cultures and controls were compared. The cultures grown on sucrose should be considerably thicker.
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The overnight cultures of the different strains were also grown on solid agar plates with and without sucrose.
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====PHOTOS====
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=Hyperspankoid characterisation=
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===Aims===
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The aim of these experiments is to ligate the promoters hyperspankoid, pspacoid and hyperspank in front of RFP of the pSB1C3 plasmid. In order to do this, the first step is to amplify the three promoter sequences and the pSB1C3 plasmid using Phusion PCR and the different primers and template DNA.
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] for Phusion PCR protocol.
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The details of the four PCR reactions are included in the table below:
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{|border=1
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|-
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!'''Tube'''
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!'''Part to be amplified'''
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!'''DNA fragment consisting the part i.e. template'''
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!'''Forward primer'''
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!'''Reverse Primer'''
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!'''Melting Temperature (Tm in °C) '''
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!'''Size of the fragment (in bp)'''
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!'''Extension time (in seconds)'''
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|-
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|1
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|Hyperspankoid
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|yneA
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|Prom_for
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|HpSpanPspacoid_rev
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|65
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|15
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|-
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|2
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|Pspacoid
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|kinA
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|Prom_for
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|HpSpanPspacoid_rev
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|65
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|15
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|-
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|3
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|Hyperspank
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|K143055 from Bacillus plate - BS022
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|Prom_for
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|Pspac-hy rev
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|62
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|15
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|-
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|4
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|Plasmid
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|vector pSB1C3/RFP
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|Vector/RFP_for
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|P2-V1
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|64
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|
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|60
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|}
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'''Table 1''': The table shows the four different Phusion PCR reactions carried out for the characterisation of the hyperspankoid promoter.
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===Results, Discussion and Conclusion===
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...
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We checked our transformants under the light microscope, expecting to see a non-filamentous phenotype since the cells had not been induced with IPTG. The resutls were as expected.
Today we checked for transformants from the filamentous Bacillus subtilis transformation (with pMutin4) we performed yesterday.
We checked our transformants under the light microscope, expecting to see a non-filamentous phenotype since the cells had not been induced with IPTG. The resutls were as expected.
"
