Team:Newcastle/19 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.


Go back to our main Lab book page

Subtilin Immunity

Aims

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.

Materials and protocol

Please refer to PCR for Phusion PCR protocol.


rocF BioBrick

Gel extraction of linearised pSB1C3

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

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