Team:Newcastle/19 August 2010

From 2010.igem.org

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===Conclusion===
===Conclusion===
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This afternoon we will be running gel electrophoresis to check the outcome of the 5 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
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Tomorrow we will be running gel electrophoresis to check the outcome of the 3 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
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Revision as of 10:54, 26 August 2010

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Contents

yneA

Gel extraction

Aims

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.

Materials and Protocol

Please refer to

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

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

We will leave the ligation overnight because concentration of yneA, pGFPrrnB and pSB1C3 are not very good. Please refer to 20.08.10 for results and discussion.

Digestion

Aim

To repeat digestion from yesterday.

Materials and Protocol

Please refer to restriction digest.


Setting up Overnight Cultures

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

Newcastle 4 Primer Tubes.jpg

Today we received the 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 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.

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. Table # below shows the different fragments, primers and conditions used for each tube.

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 68 2046 + 70
2 Promoter and RBS (pVeg-SpoVG) BioBrick Bba_K143053 Prom_for P2P2_rev 67 139 + 15
4 Double terminator pSB1AK3 consisting BBa_B0014 P1T1_for Term_rev 63 116 + 15

Table 1: 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.

  • 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.
  • The primers in bold are the new primers we received today. The primers not in bold were previously re-hydrated.

Discussion and Conclusion

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

rocF BioBrick

Gel extraction of linearised pSB1C3

Aims

We digested the plasmid pSB1C3 with the restriction enzyme EcoR1 to linearize it and then we would be running the sequence on the gel to check if the plasmid has become linear or not and then we would be extracting it.

Materials and protocol

Please refer to the:

Results

  • Lane 1: 1 Kb ladder
  • Lane 2: Linearized plasmid pSB1C3
  • Lane 3: 1 Kb ladder

There is no gel photograph because we want to keep the exposure of DNA to the UV light to an absolute minimum.

Discussion

During gel extraction procedure, we found a bright band of approximately 2072 bp size in lane 2 under UV light and we cut the gel and extracted the band.

Conclusion

We got linearized plasmid pSB1C3 and we performed gel extraction successfully and the nanodrop protocol showed that we got 22 ng/µl concentration of plasmid.

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

Aim

The aim of this experiment is to amplify plasmid linearized pSB1C3, Pspac_oid promoter and double terminator for the construction of rocF BioBrick with the help of 3 different Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:

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 P1V1 forward P2V1 reverse 65 2072 approx. 65
2 Pspacoid Promoter pMutin4 Prom_forward P2P1 reverse 67 106 approx. 15
3 Double Terminator pSB1AK3 consisting BBa_B0014 biobrick P1T1 forward Term_reverse 63 116 approx. 15

Table 2: Table represents 3 different Phusion PCR reactions for the amplification of linearized plasmid pSB1C3, Pspac_oid promoter and double treminator so that it can be ligated together with other fragments for the construction of rocF with the help of Gibson Cloning method.

  • The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
  • For more about the rocF fragments, please refer to the Cloning strategy for rocF.

Discussion

All the 3 Phusion PCR reactions were done however, gel electrophoresis will be done tomorrow, to check whether the fragments have actually amplified or not.

Conclusion

Tomorrow we will be running gel electrophoresis to check the outcome of the 3 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.

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