Latest revision as of 02:04, 28 October 2010

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Problem

Transformation of Bacillius subtilis 168 with pGFP-rrnB containing yneA

Aim

We performed gel electrophoresis on our 12 digested minipreps and found 8 were the correct product. We transformed B. subtilis 168 with one of the samples (1).

Materials and Protocol

Please refer to transformation of B. subtilis.

Results and Conclusion

Please refer to results in tomorrow's lab book.

Team:Newcastle/25 August 2010

From 2010.igem.org

Latest revision as of 02:04, 28 October 2010

Contents

Transformation of Bacillius subtilis 168 with pGFP-rrnB containing yneA

Aim

Materials and Protocol

Results and Conclusion

@@ Line 1: / Line 1: @@
 {{Team:Newcastle/mainbanner}}
-=''yneA''=
-==Gel Electrophoresis==
-===Aim===
+=Transformation of ''Bacillius subtilis'' 168 with pGFP-rrnB containing ''yneA''=
-To check if the digestion from [[Team:Newcastle/24_August_2010|yesterday]] worked.
+==Aim==
-===Materials and Protocol===
+We performed gel electrophoresis on our 12 digested minipreps and found 8 were the correct product. We transformed ''B. subtilis'' 168 with one of the samples (1).
-Please refer to [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]] for protocol.
+[[Image:filamentous_in_pgfprrnb.jpg|800px]]
-===Results===
+==Materials and Protocol==
-The result from gel electrophoresis:
-[[Image:Newcastle 25-08-2010 – Gel 1 (Ethidium Bromide).png|500px|centre]]
-'''Figure 1''' shows the double digest of 12 tubes of pGFPrrnB and ''yneA''.
-*'''Lane 1''': Vector only
-*'''Lane 2''': Tube 1
-*'''Lane 3''': Tube 2
-*'''Lane 4''': Tube 3
-*'''Lane 5''': Tube 4
-*'''Lane 6''': Tube 5
-*'''Lane 7''': Tube 6
-*'''Lane 8''': Tube 7
-*'''Lane 9''': Tube 8
-*'''Lane 10''': Tube 9
-*'''Lane 11''': Tube 10
-*'''Lane 12''': Tube 11
-*'''Lane 13''': Tube 12
-===Discussion===
-The bands we got from the gel shows that digestion in tubes 1, 2, 4, 10, 11 and 12 worked.
-===Conclusion===
-We use the digested products from the six tubes that worked for ligation.
-==Transformation of ''B. subtilis''==
-===Aim===
-To transform ''yneA'' into competent ''B. subtilis''.
-===Materials and Protocol===
 Please refer to [[Team:Newcastle/Transformation_of_B._subtilis|transformation of ''B. subtilis'']].
-===Results and Conclusion===
+==Results and Conclusion==
 Please refer to results in [[Team:Newcastle/26_August_2010|tomorrow]]'s lab book.
-==Transformation==
-===Aim===
-To transform pGFPrrnB and ''yneA'' into E. coli.
-===Materials and Protocol===
-Please refer to [[Team:Newcastle/Transformation_of_E._coli|transformation of E. coli]].
-===Results and Conclusion===
-Please refer to [[Team:Newcastle/26_August_2010|tomorrow]]'s lab book.
-==PCR==
-===Aim===
-To amplify the DNA pSB1C3 using RocF primer.
-===Materials and Protocol===
-Please refer to [[Team:Newcastle/PCR|PCR]].
-===Conclusion===
-Continue with PCR purification.
-==PCR Purification==
-===Aim===
-To remove unwanted primers, taq polymerase, buffer and salts to obtain pure DNA.
-===Materials and Protocol===
-Please refer to [[Team:Newcastle/PCR_purification|PCR purification]].
-==Digestion==
-===Aim===
-To digest the PCR products of pSB1C3 and ''yneA'' from PCR purification.
-===Materials and Protocol===
-Please refer to [[Team:Newcastle/Restriction_digests|restriction digest]].
-==Gel extraction==
-===Aim===
-To purify the DNA of ''yneA'' and pSB1C3 by extracting the bands from the gel after running gel electrophoresis. Concentration of DNA is then checked with NanoDrop.
-===Materials and Protocol===
-Please refer to:
-* [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]],
-* [[Team:Newcastle/Gel_extraction|gel extraction]] and
-* [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop spectrophotometer]].
-===Results===
-The bands we got from gel electrophoresis is very faint.
-===Conclusion===
-We realized that we used the wrong ''rocF'' primer, so we repeat the whole procedure from PCR again.
-=''rocF'' and Subtilin immunity=
-==Gel extraction==
-===Aims===
-We digested the plasmid pSB1C3 with the restriction enzyme HindIII 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:
-*[[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]],
-*[[Team:Newcastle/Gel_extraction| gel extraction]] and
-*[[TeamNewcastleNanoDrop_Spectrophotometer| NanoDrop spectrophotometer]] protocols.
-===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 approx
-During gel extraction procedure, we found a bright band of approximately 3100 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 12.7 ng/µl concentration of plasmid.
-==PCR==
-===Aim===
-The aim of this experiment is to amplify plasmid linearized pSB1C3 at 3 different melting temperatures for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of 3 different Phusion PCR.
-===Materials and Protocol===
-Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:
-===PCR===
-{|border=1
-|-
-!'''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 [[Media:Cloning_strategy_for_rocF.pdf|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.
-[[Image:Newcastle_25810gel_psb1c3.jpg|500px]]
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