Team:Newcastle/19 August 2010

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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.
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.
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 +
{|border=1
 +
|-
 +
!'''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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|'''pSB1C3_for'''
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|'''pSB1C3_rev'''
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|53.3
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|2046 +
 +
|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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|51.7
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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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|50.9
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|116 +
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|15
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|}
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'''Table 5''': This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts 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.
 +
* Please note: The newly arrived primer tubes have to be handled with extra caution, because they will be the main stocks which working stock solutions will be made from. Therefore, gloves have to be worn, as well as preventing any contamination. Water will be used in order to liquefy the primers and the water used will be from Pure Lab Distilled Water.
=''rocF'' BioBrick=
=''rocF'' BioBrick=

Revision as of 08:29, 20 August 2010

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Contents

Gel extraction of yneA, pGFPrrnB and pSB1C3

Aims

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.

Materials and Protocol

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

Results

yneA 1 yneA 2 yneA 3 pSB1C3 pGFPrrnB
Concentration of DNA ng/µl 3.4 4.7 5.4 2.2 18.5

Discussion

The bands we extracted from the gel was good for pGFPrrnB, but not so strong for yneA and PSB1C3.

The results we got from the nanodrop were not as good as expected for yneA and PSB1C3.

Conclusion

We will proceed with digestion, but another set of ligation will be set up to repeat the process again.

Ligation for pGFPrrnB with yneA and pSB1C3 with yneA

Aims

To ligate yneA into both vectors pGFPrrnB and pSB1C3.

Materials and Protocol

Please refer to ligation.

Ligation mix for pSB1C3 with yneA

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.

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

Results and Conclusion

Please refer to 20.08.10.


Digestion of yneA, pGFPrrnB and pSB1C3

Aim

To repeat digestion from 18.08.10.

Materials and Protocol

Please refer to restriction digest.


Setting up Overnight Cultures for miniprep

Aims

To prepare cultures for miniprep of yneA, pGFPrrnB and pSB1C3 tomorrow.

Materials and Protocol

Please refer to growing an overnight culture.



Subtilin Immunity

Aims

Today we received four new primers that we ordered:

  • Prom_For
  • pSB1C3_for
  • pSB1C3_rev
  • Term_rev

These primers were ordered as a solution to the problem we encountered when trying to carry out the Gibson protocol for the Subtilin Immunity (and RocF) BioBrick. These 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.

Materials and protocol

Please refer to DNA rehydration page for the protocol for the rehydration of the four primers and to the Phusion PCR page for protocol followed for Phusion PCR.

Tube Part to be amplified DNA fragment consisting the part Forward primer Reverse Primer Melting Temperature (Tm in °C) Size of the fragment (in bp) Extension time* (in seconds)
1 Plasmid Vector pSB1C3 pSB1C3_for pSB1C3_rev 53.3 2046 + 70
2 Promoter and RBS (pVeg-SpoVG) BioBrick Bba_K143053 Prom_for P2P2_rev 51.7 139 + 15
4 Double terminator pSB1AK3 consisting BBa_B0014 P1T1_for Term_rev 50.9 116 + 15

Table 5: This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.

  • 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.
  • Please note: The newly arrived primer tubes have to be handled with extra caution, because they will be the main stocks which working stock solutions will be made from. Therefore, gloves have to be worn, as well as preventing any contamination. Water will be used in order to liquefy the primers and the water used will be from Pure Lab Distilled Water.

rocF BioBrick

Gel extraction of linearised pSB1C3

PCR of pSB1C3, pspac oid and double terminator with new primers

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