From 2009.igem.org

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Latest revision as of 20:00, 21 October 2009

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Lab Notebook

Week Beginning: 20th July 2009
	Experiments	Conclusion
Monday	No experiments were undertaken	The aim of the week was to become acquainted with the protocols.
Tuesday	No experiments were undertaken
Wednesday	Pure Yield Plasmid Prep: practice session
Thursday	No experiments were undertaken
Friday	DNA Gel Electrophoresis: practice session

Week Beginning: 27th July 2009
	Experiments	Conclusion
Monday	Bio brick elution and transformation	Experiments were carried out to become acquainted with the protocols.
Tuesday	Double digestion protocol
Wednesday	Gel extraction and purification
Thursday	High fidelity PCR
Friday	No experiments were undertaken

Week Beginning: 3rd Aug 2009
	Experiments	Conclusion
Monday	No experiments undertaken	The aim of the week was to start transforming the necessary biobricks needed for the project.
Tuesday	Biobrick DNA Elution and Transformation: E0040 K145270 C0012 I719005 R0071 K084009 C0161 K091100
Wednesday	Plasmid Prep (using transformed biobricks prepared yesterday – except C0161) DNA Elution and Transformation C0178 K081008 C0179 K091146 C0062 K091107 C0161
Thursday	Plasmid Prep (using biobricks transformed yesterday)
Friday	Repeat Biobrick transformations: C0161

Week Beginning: 10th August 2009
	Experiments	Conclusion
Monday	Culture overnight of transformed parts B0015-Failed B0034 –Failed J23119-Failed C0161-Failed R0062 C0170 C0171 R0079	The main aim of this week was to grow up any bio-bricks that may be required. Several parts failed and so had to be repeated. There are many reasons for the failure, one being the use of the wrong cells and transformations failing.
Tuesday	Plasmid Maxiprep of the overnight cultures
Wednesday	Culture overnight of transformed parts C0161 B0034 B0015 J23119 R0010 R0011
Thursday	Plasmid maxiprep of the overnight cultures
Friday	Digestion of parts for Quality control

Week Beginning: 17th Aug 2009
	Experiments	Conclusion
Monday	No experiments were undertaken	The aim of the week was to start ligating biobrick parts together. The aim for next week is to check that the ligations have been successful.
Tuesday	Digestion of parts for quality control. Polymerase Chain Reaction: C0012 C0170 C0171 C0179 C0161 C0178
Wednesday	Polymerase Chain Reaction: the reaction from yesterday failed hence the PCR was repeated Starter Culture and Maxi Prep: mCherry mBanana GFP
Thursday	Double Digestion of PCR samples: C0012 C0171 C0170 C0179 C0161 C0178 PCR Purification
Friday	Ligation of Biobrick parts- all the following parts were ligated with B0034: C0171 C0179 C0012 C0178 C0170 C0062 C0161

Week Beginning: 24th August 2009
	Experiments	Conclusion
Monday	Ligation of B0034 with C0178, C0062, E0040, C0171, C0179, C0170, C0012, C0161, E0040	The main aim of this week was to get our first ligations to work, however this wasn’t the case and so after the ligations were repeated and failed again, troubleshooting began.
Tuesday	Transformation of B0034 with biobrick parts: C0178, C0062, E0040, C0171, C0179, C0170, C0012, C0161, E0040
Wednesday	Colony PCR of ligated parts FAILED PCR of Ptac from Pmal vector, which introduces prefix and suffix followed by a double digestion of pTAC and RBS.
Thursday	Repeat of Colony PCR, which had previously failed.
Friday	PCR of biobrick inserts C0178, C0062, E0040, C0171, C0179, C0170, C0012, C0161, E0040 Required due to previous failure using up stocks.

Week Beginning: 31st Aug 2009
	Experiments	Conclusion
Monday	No experiments were done today (Bank Holiday)	The aim of this week was to continue trying to get our first ligations to work, this wasn’t successful the digestion was found not to be complete and so this is thought to be one of the reasons behind the failed ligations. The next set of Digestions will be done overnight instead of the usual 2 hours.
Tuesday	Repeat of Colony PCR of: pTac – B0034 DNA Double Digestion: (pTac – B0034) Ligation: (pTac – B0034)
Wednesday	Colony PCR Control: Method as per 28/08 but both B0034 and pTac colonies were diluted in 100μl. Single Digestion of biobricks: single digestion was performed as it was discovered that one of the enzymes was not working efficiently. C0171 C0179 C0012 C0178 C0170 C0062 C0161 E0040 B0034 DNA Transformation: pTac and B0034 Culture of C0161, pTac –B0034 and B0034
Thursday	Mini Prep C0161 B0034-A B0034-B
Friday	Second Single Digestion: C0171 C0170 C0012 C0179 E0040 0034 Biobricks (C0178, C0062, C0161 were transformed again)

Week Beginning: 7th Sept 2009
	Experiments	Conclusion
Monday	PCR of Pmal to produce Ptac Purification Gel of C0171, C0012, C0170, C0179, E0040, B0034. B0034 was treated with SAP. C0171, C0012 and C0170 was ligated to B0034	TSAP (thermo sensitive alkaline phosphatase) was used on the plasmid to dephosphorylate the plasmid so it cannot religate. Digestions with PstI were done for 8 hours and every other digestion was run for 3 hours. Ligations are now ran overnight at 4C.
Tuesday	Digestion of Ptac with EcoRI and XbaI and RBS with XbaL and Spel RBS was treated with SAP Ligation of Ptac to RBS Culture overnight of biobrick parts C0161, C0178 and C0062 as these were shown to be dodgy parts.
Wednesday	A maxiprep was performed on the parts, which were previously cultured overnight. Digestion of the parts with Pstl to check that the parts were correct.
Thursday	PCR of C0161, C0178, C0062 which didn’t work
Friday	PCR of Gfp-LVA, which didn’t work.

Week Beginning: 14th Sept 2009
	Experiments	Conclusion
Monday	Transformation of C0170, C0012 and pTac to RBS PCR of GFP-LVA with different primers and it worked.	The ligations of Ptac didn’t work with the 3-hour digestion and TA cloning was inconclusive, due to a lack of resources. Parts, which were shown to be dodgy, were re-grown and checked for quality control.
Tuesday	Colony PCR of the ligated parts Culture overnight of C0161, C0178, C0062.
Wednesday	Digestion of Biobricks pLux, STOP and LacI.followed by an overnight ligation of the parts TA cloning of Ptac. Maxiprep of the cultures from previous day.
Thursday	Transformation of the ligated parts PCR of GFP LVA suffix with different primers, which was successful again.
Friday	Colony PCR of the ligated parts PCR of C0161, C0178 that worked.

Week Beginning: 21st Sept 2009
	Experiments	Conclusion
Monday	Diluted colony PCR of the C0012-B0015, R0062-C0012. Overnight digestion of Ptac. Ligation of Gfp-LVA to plasmid.	TA cloning was performed on Gfp-LVA to check the prefix and the suffix was digested properly. Ptac also had a successful TA cloned vector. This means that the work to check for complete digestion can begin. The main focus of this week was to finish any wiki work as this is the last week for the iGEM students.
Tuesday	PCR of Ptac from Pmal Overnight ligation for TA cloning. Transformation of previously ligated part.
Wednesday	Digestion of C0012 and C0179 Gel extraction was performed, yielding too low concentrations for a ligation to occur.
Thursday	Repeat of the previous day’s experiment followed by ligation overnight. Culture of the Gfp which was TA cloned
Friday	Maxiprep of the cultured Gfp. This failed as the DNA was sheared.

Week Beginning: 28th Sept 2009
	Experiments	Conclusion
Monday	Culture of the TA cloned Ptac Transformation of C0012-C0179 and Ptac-RBS	The transformations failed from the beginning of the week and the conclusion of the Gfp after ligation wasn’t reached until the next week.
Tuesday	The Ptac and Gfp-LVA which was TA cloned was digested overnight. Colony PCR was performed on the ligations and was shown to have failed.
Wednesday	A gel was run on the Ptac and was shown to have digested improperly. Gel purification was performed on Gfp-LVA
Thursday	Digestion of Ptac overnight and further purification of GFP-LVA.
Friday	Ligations of Ptac and Gfp-LVA with PSB1A3.

Week Beginning: 5th Oct 2009
	Experiments	Conclusion
Monday	Digestion of Ptac with EcoRI HF and SpeI Ptac Gel purification TSAP with PSB1A3 ES digests. Ligation of GFP-LVA (ES) into PSB1A3 Transformation of previous Ligation More plates made up	Finally there was a successful ligation. The conditions, which seemed to work, were 18 hours at 16°C. Work is now going to be focussed on showing the functionality of the Ptac.
Tuesday	No colonies grew on the plates, suspected that the T4 Ligase was improperly killed.
Wednesday	Ligation of Ptac and GFP-LVA with PSB1A3
Thursday	Transformations performed on the previous ligations
Friday	Colonies observed on plates. Colony PCR performed on both, GFP-LVA discovered not to work. Ptac, all colonies picked were successful

Week Beginning: 12th Oct 2009
	Experiments	Conclusion
Monday	Grew up Ptac colonies, 3x Minipreps were performed. The yield was very low, so a culture for a maxiprep was then put on. Digestion of RBS, GFP and B0015.	The focus of this week was to build a construct, which could prove the functionality of the Ptac part.
Tuesday	Ptac was Maxiprep’d. The yield again was very low, we think this was due to the introduction of the insert causing the plasmid to become low copy. Gel purification of the previously digested parts.
Wednesday	Single Digestion of Ptac was performed.
Thursday	After gel purification of the Ptac, the yield was very low and so a double digestion of Ptac was performed.
Friday	Gel purification of Ptac part. TSAP of the E0040 vector. Ligation of Ptac and E0040.

Week Beginning: 17th Oct 2009
	Experiments	Conclusion
Saturday	Transformation of the previously ligated parts. Double digestion of the TA cloned Ptac.	The focus on this week was to continue trying to build a part, which will show functionality, and to finish off everything ready for the jamboree.
Sunday	Gel purification of the Ptac followed by ligation to the E0040 vector. Colonies were observed, however after colony PCR, the ligation was shown to have failed.
Monday	Cultures of a fluorescent protein were grown up with the Ptac inserted.
Tuesday	Fluorescence studies were conducted on the Ptac cultures. Last minute touches to the Wiki, Poster and presentation.
Wednesday	DEADLINE FOR WIKI FREEZE

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 <li class=" cssMenui"><a class="  cssMenui" href="https://2009.igem.org/Team:Southampton/Project/Intro">Project Introduction</a></li>
 <li class=" cssMenui"><a class="  cssMenui" href="https://2009.igem.org/Team:Southampton/Project">Project Description</a></li>
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 			<li class=" cssMenui"><a class="  cssMenui" href="https://2009.igem.org/Team:Southampton/Project/Protocols/PlasmidPrep">Plasmid Preparation</a></li>
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Team:Southampton/Notebook/Labbook

From 2009.igem.org

Latest revision as of 20:00, 21 October 2009

Lab Notebook

Week Beginning: 20th July 2009

Experiments

Conclusion

Monday

No experiments were undertaken

The aim of the week was to become acquainted with the protocols.

Tuesday

No experiments were undertaken

Wednesday

Pure Yield Plasmid Prep: practice session

Thursday

No experiments were undertaken

Friday

DNA Gel Electrophoresis: practice session

Week Beginning: 27th July 2009

Experiments

Conclusion

Monday

Bio brick elution and transformation

Experiments were carried out to become acquainted with the protocols.

Tuesday

Double digestion protocol

Wednesday

Gel extraction and purification

Thursday

High fidelity PCR

Friday

No experiments were undertaken

Week Beginning: 3rd Aug 2009

Experiments

Conclusion

Monday

No experiments undertaken

The aim of the week was to start transforming the necessary biobricks needed for the project.

Tuesday

Biobrick DNA Elution and Transformation:

E0040 K145270 C0012 I719005 R0071 K084009 C0161 K091100

Wednesday

Plasmid Prep (using transformed biobricks prepared yesterday – except C0161)

DNA Elution and Transformation C0178 K081008 C0179 K091146 C0062 K091107 C0161

Thursday

Plasmid Prep (using biobricks transformed yesterday)

Friday

Repeat Biobrick transformations: C0161

Week Beginning: 10th August 2009

Experiments

Conclusion

Monday

Culture overnight of transformed parts

B0015-Failed B0034 –Failed J23119-Failed C0161-Failed R0062 C0170 C0171 R0079

The main aim of this week was to grow up any bio-bricks that may be required. Several parts failed and so had to be repeated. There are many reasons for the failure, one being the use of the wrong cells and transformations failing.

Tuesday

Plasmid Maxiprep of the overnight cultures

Wednesday

Culture overnight of transformed parts

C0161 B0034 B0015 J23119 R0010 R0011

Thursday

Plasmid maxiprep of the overnight cultures

Friday

Digestion of parts for Quality control

Week Beginning: 17th Aug 2009

Experiments

Conclusion

Monday

No experiments were undertaken

The aim of the week was to start ligating biobrick parts together. The aim for next week is to check that the ligations have been successful.

Tuesday

Digestion of parts for quality control.

Polymerase Chain Reaction:

C0012 C0170 C0171 C0179 C0161 C0178

Wednesday

Polymerase Chain Reaction: the reaction from yesterday failed hence the PCR was repeated

Starter Culture and Maxi Prep: mCherry mBanana GFP

Thursday

Double Digestion of PCR samples: C0012 C0171 C0170 C0179 C0161 C0178

PCR Purification

DNA Elution and Transformation
C0178 K081008 C0179 K091146 C0062 K091107 C0161

Repeat Biobrick transformations:
C0161

Starter Culture and Maxi Prep:
mCherry mBanana GFP

Double Digestion of PCR samples:
C0012 C0171 C0170 C0179 C0161 C0178

Transformation of B0034
with biobrick parts:
C0178, C0062, E0040, C0171, C0179, C0170, C0012, C0161, E0040

Colony PCR of ligated parts
FAILED

C0178, C0062, E0040, C0171, C0179, C0170, C0012, C0161, E0040
Required due to previous failure using up stocks.

No experiments were done today
(Bank Holiday)

Colony PCR Control:
Method as per 28/08 but both B0034 and pTac colonies were diluted in 100μl.

DNA Transformation: pTac and B0034
Culture of C0161, pTac –B0034 and B0034

Mini Prep
C0161 B0034-A B0034-B

C0171 C0170 C0012 C0179 E0040 0034
Biobricks (C0178, C0062, C0161 were transformed again)

PCR of Pmal to produce Ptac
Purification Gel of C0171, C0012, C0170, C0179, E0040, B0034. B0034 was treated with SAP.
C0171, C0012 and C0170 was ligated to B0034

Digestion of Ptac with EcoRI and XbaI and RBS with XbaL and Spel RBS was treated with SAP
Ligation of Ptac to RBS
Culture overnight of biobrick parts C0161, C0178 and C0062 as these were shown to be dodgy parts.

Transformation of C0170, C0012 and pTac to RBS
PCR of GFP-LVA with different primers and it worked.