=Gel extraction and ligation of filamentous cell part into pGFPrrnB and pSB1C3=
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==Gel extraction==
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==Aims==
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===Aims===
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The aim of the experiment is to further purify the filamentous cell part (''yneA''), pGFPrrnB and pSB1C3 by doing gel extraction. The purified fragments are then ligated together.
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To purify the products of ''yneA'', pGFPrrnB and PSB1AT3 by extracting the bands from the gel after running gel electrophoresis. Concentration of the DNA and vectors are then checked with nanodrop.
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===Materials and Protocol===
===Materials and Protocol===
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Please refer to
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Please refer to [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]], [[Team:Newcastle/Gel_extraction|gel extraction]], [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop spectrophotometer]] and [[Team:Newcastle/Ligation|ligation]].
The bands we extracted from the gel was good for pGFPrrnB, but not so strong for ''yneA'' and PSB1C3.
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The results we got from the nanodrop were not as good as expected for ''yneA'' and PSB1C3.
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===Conclusion===
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We will proceed with digestion, but another set of ligation will be set up to repeat the process again.
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==Ligation==
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===Aims===
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To ligate ''yneA'' into both vectors pGFPrrnB and pSB1C3.
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/Ligation|ligation]].
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====Ligation mix for pSB1C3 with ''yneA''====
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The concentration of pSB1C3 and ''yneA'' that we got from the gel extraction earlier today was very low, so we used a different mixture set up for ligation.
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Ligation mix:
Ligation mix:
Line 100:
Line 51:
|}
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===Results and Conclusion===
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'''Table 1''': Ligation mix for ligation of ''yneA'' into pGFPrrnB and pSB1C3
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We will leave the ligation overnight because concentration of ''yneA'', pGFPrrnB and pSB1C3 are not very good. Please refer to [[Team:Newcastle/20_August_2010|20.08.10]] for results and discussion.
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===Results===
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==Digestion==
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===Aim===
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To repeat digestion from [[Team:Newcastle/18_August_2010|yesterday]].
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/Restriction_digests|restriction digest]].
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==Setting up Overnight Cultures==
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===Aims===
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To prepare cultures for miniprep of ''yneA'', pGFPrrnB and pSB1C3 [[Team:Newcastle/20_August_2010|tomorrow]].
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/Growing_an_overnight_cultures|growing an overnight culture]].
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=Subtilin Immunity=
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==Aims==
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[[Image:Newcastle 4 Primer Tubes.jpg|150px|right]]
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Today we received the four new primers that we ordered:
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*Prom_For
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*pSB1C3_for
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*pSB1C3_rev
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*Term_rev
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These primers were ordered as a solution to the problem we encountered before we were about to attempt carry out the Gibson protocol for the Subtilin Immunity (and RocF) BioBrick. The primers will be used with previously rehydrated primers to try and obtain all four parts that we need to make the Subtilin Immunity BioBrick using the Gibson Protocol. So far the ''spaIFEG'' coding sequence (part 3) has already been successfully obtained. We still need to obtain the plasmid vector (part 1), promoter & RBS (part 2) and double terminator (part 4). So in order to do this the primers received today will be rehydrated and Phusion PCR will be carried out for each of the parts in order to amplify these parts.
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==Materials and protocol==
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Please refer to [[Team:Newcastle/DNA_re-hydration| DNA rehydration]] page for the protocol for the rehydration of the four primers and to the [[Team:Newcastle/PCR| Phusion PCR]] page for protocol followed for Phusion PCR. Table # below shows the different fragments, primers and conditions used for each tube.
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{|border=1
{|border=1
|-
|-
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!'''Tube'''
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!
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!'''Part to be amplified'''
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!''yneA'' 1
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!'''DNA fragment consisting the part'''
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!''yneA'' 2
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!'''Forward primer'''
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!''yneA'' 3
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!'''Reverse Primer'''
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!pSB1C3
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!'''Melting Temperature (Tm in °C) '''
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!pGFPrrnB
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!'''Size of the fragment (in bp)'''
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|-
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!'''Extension time* (in seconds)'''
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!Concentration of DNA ng/µl
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|-
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!3.4
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|1
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!4.7
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|Plasmid Vector
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!5.4
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|pSB1C3
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!2.2
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|pSB1C3_for
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!18.5
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|pSB1C3_rev
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|68
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|2046 +
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|70
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|-
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|2
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|Promoter and RBS (pVeg-SpoVG)
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|BioBrick Bba_K143053
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|'''Prom_for'''
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|P2P2_rev
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|67
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|139 +
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|15
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|-
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|4
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|Double terminator
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|pSB1AK3 consisting BBa_B0014
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|P1T1_for
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|'''Term_rev'''
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|63
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|116 +
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|15
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|}
|}
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'''Table 1''': This table shows the three different Phusion PCR reactions that were carried out today. If this is successful, these three parts (part 1, 2 and 4) along with (part 3) which is already obtained can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.
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'''Table 2''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
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*The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
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*The primers in bold are the '''new primers''' we received today. The primers not in bold were previously re-hydrated.
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== Discussion and Conclusion ==
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Tommorrow we will carry out a test gel to check the sizes of the amplified fragments and if the sizes are correct then gel extraction will be performed
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=''rocF'' BioBrick=
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==Gel extraction of linearised pSB1C3==
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===Aims===
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We digested the plasmid pSB1C3 with the restriction enzyme EcoR1 to linearize it and then we would be running the sequence on the gel to check if the plasmid has become linear or not and then we would be extracting it.
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===Materials and protocol===
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Please refer to the:
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*[[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]],
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*[[Team:Newcastle/Gel_extraction| gel extraction]] and
There is no gel photograph because we want to keep the exposure of DNA to the UV light to an absolute minimum.
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===Discussion===
===Discussion===
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During gel extraction procedure, we found a bright band of approximately 2072 bp size in lane 2 under UV light and we cut the gel and extracted the band.
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The DNA concentration obtained for ''yneA'' ranged from 3.4 µl/ml to 5.4 µl/ml, 2.2 µl/ml for pSB1C3 and 18.5 µl/ml for pGFPrrnB . Therefore the concentration for ''yneA'' and pSB1C3 is low.
===Conclusion===
===Conclusion===
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We got linearized plasmid pSB1C3 and we performed gel extraction successfully and the nanodrop protocol showed that we got 22 ng/µl concentration of plasmid.
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We will proceed with digestion, but another set of ligation will be set up to repeat the process again.
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==PCR of pSB1C3, pspac oid and double terminator with new primers==
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===Results and Conclusion===
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===Aim===
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The aim of this experiment is to amplify plasmid linearized pSB1C3, Pspac_oid promoter and double terminator for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of 3 different Phusion PCR.
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===Materials and Protocol===
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Ligation will be done overnight because concentration of the filamentous cell part, pGFPrrnB and pSB1C3 are not very good. Please refer to [[Team:Newcastle/20_August_2010|20.08.10]] for results and discussion. Overnight culture of ''yneA'', pSB1C3 and pGFPrrnB were set up for miniprep extraction tomorrow, 20th August, 2010.
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Please refer to [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 5 PCR reactions are mentioned below:
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===PCR===
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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'''
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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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|Plasmid Vector
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|pSB1C3
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|P1V1 forward
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|P2V1 reverse
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|58
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|2072 approx.
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|60
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|-
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|2
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|Pspacoid Promoter
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|pMutin4
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|P1P1 forward
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|P2P1 reverse
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|49
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|106 approx.
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|15
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|-
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|3
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|1st fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P1S1 forward
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|P2S1 reverse
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|58
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|246 approx.
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|15
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|-
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|4
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|2nd fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P3S2 forward
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|P4S2 reverse
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|65
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|597 approx.
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|20
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|-
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|5
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|3rd fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P5S3 forward
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|P6S3 reverse
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|66
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|125 approx.
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|15
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|-
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|6
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|Double Terminator
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|pSB1AK3 consisting BBa_B0014 biobrick
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|P1T1 forward
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|P2T1 reverse
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|56
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|116 approx.
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|15
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|}
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'''Table 1''': Table represents 5 different Phusion PCR reactions for the amplification of linearized plasmid pSB1C3, so that it can be ligated together with other fragments for the construction of ''rocF'' with the help of Gibson Cloning method.
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*For the 3rd PCR reaction where the melting temperature is set for 65°C, the number of cycles were increased to 35 instead of 29.
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* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
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* For more about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
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===Discussion===
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All the 5 Phusion PCR reactions were done however, gel electrophoresis will be done tomorrow, to check whether the fragments have actually amplified or not.
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===Conclusion===
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This afternoon we will be running gel electrophoresis to check the outcome of the 5 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
Gel extraction and ligation of filamentous cell part into pGFPrrnB and pSB1C3
Aims
The aim of the experiment is to further purify the filamentous cell part (yneA), pGFPrrnB and pSB1C3 by doing gel extraction. The purified fragments are then ligated together.
Table 1: Ligation mix for ligation of yneA into pGFPrrnB and pSB1C3
Results
yneA 1
yneA 2
yneA 3
pSB1C3
pGFPrrnB
Concentration of DNA ng/µl
3.4
4.7
5.4
2.2
18.5
Table 2: Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
Discussion
The DNA concentration obtained for yneA ranged from 3.4 µl/ml to 5.4 µl/ml, 2.2 µl/ml for pSB1C3 and 18.5 µl/ml for pGFPrrnB . Therefore the concentration for yneA and pSB1C3 is low.
Conclusion
We will proceed with digestion, but another set of ligation will be set up to repeat the process again.
Results and Conclusion
Ligation will be done overnight because concentration of the filamentous cell part, pGFPrrnB and pSB1C3 are not very good. Please refer to 20.08.10 for results and discussion. Overnight culture of yneA, pSB1C3 and pGFPrrnB were set up for miniprep extraction tomorrow, 20th August, 2010.
"
