Team:Newcastle/19 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=Gel extraction of ''yneA''=
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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.
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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_extraction|gel extraction]].
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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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Ligation mix:
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{|border=1
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|-
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!'''Reagents'''
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!'''1:3(μl)'''
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!'''1:5(μl)'''
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!'''Vector(μl)'''
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|-
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|'''Vector'''
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|3
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|2
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|1.5
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|-
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|'''Insert'''
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|3
 +
|6
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|7.5
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|-
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|'''10X BUFFER'''
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|1
 +
|1
 +
|1.1
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|-
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|'''T4 Ligase'''
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|1
 +
|1
 +
|1
 +
|-
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|'''H2O'''
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|2
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|0
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|0
 +
|-
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|'''Total Volume'''
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|10.0
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|10.0
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|10.0
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|}
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'''Table 1''': Ligation mix for ligation of ''yneA'' into pGFPrrnB and pSB1C3
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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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'''Table 2''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
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===Discussion===
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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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===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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===Results and Conclusion===
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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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'''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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