Team:Newcastle/19 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=Gel extraction of ''yneA'', pGFPrrnB and pSB1C3=
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=Gel extraction and ligation of filamentous cell part into pGFPrrnB and pSB1C3=
==Aims==
==Aims==
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To extract the correct size bands for ''yneA'', pGFPrrnB and PSB1AT3 from the gel after running gel electrophoresis. Concentration of the DNA and vectors are also checked with nanodrop.
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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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==Materials and Protocol==
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===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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!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 for pGFPrrnB with ''yneA'' and pSB1C3 with ''yneA''=
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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 of ''yneA'', pGFPrrnB and pSB1C3=
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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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=Setting up Overnight Cultures for miniprep=
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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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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
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=Subtilin Immunity=
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==Aims==
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We have modified the promoters for Plasmid Vector (part 1), Promoter & RBS (part 2) and Double terminator (part 4), thus we aim to do Phusion PCR today.  
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==Materials and protocol==
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===Results and Conclusion===
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Please refer to [[Team:Newcastle/PCR| PCR]] for Phusion PCR 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.
{{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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