Team:Newcastle/19 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=''yneA''=
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=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 [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]], [[Team:Newcastle/Gel_extraction|gel extraction]], [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop spectrophotometer]].
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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]].
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===Results===
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{|border=1
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|-
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!
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!''yneA'' 1
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!''yneA'' 2
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!''yneA'' 3
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!pSB1C3
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!pGFPrrnB
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|-
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!Concentration of DNA ng/µl
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!3.4
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!4.7
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!5.4
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!2.2
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!18.5
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|}
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===Discussion===
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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:
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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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Please refer to [[Team:Newcastle/20_August_2010|20.08.10]].
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===Results===
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{|border=1
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|-
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!
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!''yneA'' 1
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!''yneA'' 2
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!''yneA'' 3
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!pSB1C3
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!pGFPrrnB
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|-
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!Concentration of DNA ng/µl
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!3.4
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!4.7
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!5.4
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!2.2
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!18.5
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|}
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==Digestion==
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'''Table 2''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
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===Aim===
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===Discussion===
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To repeat digestion from [[Team:Newcastle/18_August_2010|18.08.10]].
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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.
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===Materials and Protocol===
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===Conclusion===
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Please refer to [[Team:Newcastle/Restriction_digests|restriction digest]].
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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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===Results and Conclusion===
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==Setting up Overnight Cultures==
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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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===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
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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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|1
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|Plasmid Vector
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|pSB1C3
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|'''pSB1C3_for'''
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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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|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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|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 #''': 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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*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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==PCR of pSB1C3, pspac oid and double terminator with new primers==
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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
 
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 00:19, 28 October 2010

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Contents

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.

Materials and Protocol

Please refer to gel electrophoresis, gel extraction, nanodrop spectrophotometer and ligation.

Ligation mix:

Reagents 1:3(μl) 1:5(μl) Vector(μl)
Vector 3 2 1.5
Insert 3 6 7.5
10X BUFFER 1 1 1.1
T4 Ligase 1 1 1
H2O 2 0 0
Total Volume 10.0 10.0 10.0

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.


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