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Meetings Calendar

Formal Lab Session - 21st September 2009

Stochastic switch team

Today we digested the sac minipreps with EcoRI and XbaI. It seems clear that there was something wrong with the minipreps (such as poor procedure) as there seemed to be little or no DNA in the sample.

Did a gel fragment clean up on the sspb fragment cut from the gel, this was put in the 'fragments' box in the freezer ready for later.

50ul of ara+pSB1AT3 plasmid was cut with SpeI and PstI in order to insert the sspb fragment after the operator. Rather than doing a gel fragment prep a PCR clean up (kit) was done on the digest to save time.

We ligated the ara + sspb fragments using Chris' method:

2ul Ligase buffer
2.5ul Vector DNA
14.5ul insert DNA
1ul ligase

= 20ul

This was left in the fridge overnight to improve the ligation conditions.

Metal Sensing Team

Introduction

Practical Outline

Here are the list of tasks which need to be accomplished by the end of the day:

Run analysis digests of the 5 midi-preps of cotC, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3 (midi-prepped in the 11/09/09 lab session and digested in the 18/09/09 lab session)
1. If successful, double digest 40ul of pMUTIN4 with BamHI and HindIII and place under DNA gel electrophoresis; excise band produced and extract DNA
2. If unsuccessful, inoculate 5ml of LB with E. coli containing pMUTIN4 (for mini-preps and midi-preps)
Check PCR reaction (amplification of cotC-GFP-smtA) on gel and clean up if successful
1. If successful, digest with BamHI and HindIII
Should the pMUTIN4 DNA extraction be a success and the cotC-GFP-smtA BioBrick be successfully amplified (by PCR) and extracted, ligate the two fragments together.
1. Reactions include:
  1. vector + insert + ligase
  2. vector + ligase
  3. vector and NO ligase
Inoculate 3ml of LB with TPA2 cells (for production of competent DH5-alpha E. coli cells)

Procedure

Analysing midi-prep digests

Team Newcastle 2009 iGEM Geldoc 2009-09-21 13hr 11min.jpg

Double digesting pMUTIN4 midi-prep DNA

Cleaning up cotC PCR product

Excising digested pMUTIN4 from gel

Quantification of cotC and pMUTIN4

Chassis team

Introduction

We have already got the double clone,the next thing need to do is to ligate this sleB:cwlJ fragment with pMutin4 plasmid to get the pspac sequence from pMutin4. pMutin4 contains HindIII and BamHI restriction sites, but our double clone sequence didn't contain those sites. If we want to ligate double clone fragment with pMutin4 backbone, we need PCR our double clone fragment and add HindIII and BamHI restriction sites.We designed cwlJsleB primers here.
We digest pMutin4 with HindIII and BamHI to get our plasmid backbone.
Midi Prep L1 and L2 transformation strains to get large amount of plasmid.
Our kinA synthesized part was arrived, we need to construct this gene into our integrate plasmid and transform it into B.subtilis.

Experiment procedure

PCR doulbe clone fragment

Prepare buffer mix and Taq

 Reaction buffer  10ul x 4 = 40ul
            dNTP   2ul x 4 =  8ul
 --------------------------------
                             48ul
 12ul for each reaction

 GoTaq(5U/ul)   0.25ul x 2 =  0.5ul
 ddH2O                        4.5ul
 ----------------------------------
                                5ul
 2.5ul for each reaction

Exp. PCR experiment for gene cwlJ:sleB
No.	Water (ul)	PCR Mix (ul)	Forward Primer (ul)	Reverse Primer (ul)	Template (ul)	Polymerase used (ul)	Total Volume (ul)
1	28.5	12	cwlJsleBForward 2.5ul	cwlJsleBReverse 2.5ul	pSB1AT3:cwlJ:sleB 2ul	GoTaq 2.5ul	50
2	30.5	12	cwlJsleBForward 2.5ul	cwlJsleBReverse 2.5ul	0ul	GoTaq 2.5ul	50

PCR procedure: STD50

95C 2min -->[95C 30S --> 50C 30S --> 75C 1min] 30 runs --> 75C 5min --> store at 4C

Midi Prep

Before we carried out Midi Prep, we take out 1ml of cell mix and put them into 1.5ml eppendorf tube to keep in fridge.
50ml of cells were used for Midi Prep.
Midi prep processed by following the standard protocal of Plasmid Midi Prep Kit(Sigma).

Digest pMutin4 plasmid for ligation

Prepare the digest reaction.

 dd H2O                     7ul
 10X fast digest buffer     7ul
 Fast HindIII               3ul
 Fast BamHI                 3ul
 pMutin4 plasmid           50ul
 ------------------------------
                           70ul

Incubate 1 hour at 37 degree.

Digest kinA fragment

Digest pMK-RQ vector to get kinA fragment.(Jess has done this part)
Run big well gel to seperate kinA and pMK-RQ backbone.
Use gel extraction kit to purify kinA fragment.

Conclusion

When we cut pMK-RQ with EcoRI and NheI, both pMK-RQ back bone and kinA fragment are about 2k bp. To get the purified kinA fragment, we need to run the gel for a longer time to seperate the two bands. The other way is to introduce the third enzyme which can cut the backbone into 2 pieces and keep kinA's integrability.

Futher plan

Ligate kinA with pGFP-rrnB.
Ligate cwlJ:sleB fragment with pMutin4 and transform ligated plasmid into E.coli.

July
M	T	W	T	F	S	S
		[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_July_2009&action=edit 1]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_July_2009&action=edit 2]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_July_2009&action=edit 3]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_July_2009&action=edit 4]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_July_2009&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_July_2009&action=edit 6]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/7_July_2009&action=edit 7]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_July_2009&action=edit 8]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_July_2009&action=edit 9]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_July_2009&action=edit 10]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_July_2009&action=edit 11]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_July_2009&action=edit 12]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_July_2009&action=edit 13]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/14_July_2009&action=edit 14]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_July_2009&action=edit 15]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_July_2009&action=edit 16]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_July_2009&action=edit 17]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_July_2009&action=edit 18]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_July_2009&action=edit 19]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_July_2009&action=edit 20]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_July_2009&action=edit 21]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_July_2009&action=edit 22]	[http://2009.igem.org/Team:Newcastle/Labwork/23_July_2009 23]	[http://2009.igem.org/Team:Newcastle/Labwork/24_July_2009 24]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_July_2009&action=edit 25]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_July_2009&action=edit 26]
[http://2009.igem.org/Team:Newcastle/Labwork/27_July_2009 27]	[http://2009.igem.org/Team:Newcastle/Labwork/28_July_2009 28]	[http://2009.igem.org/Team:Newcastle/Labwork/29_July_2009 29]	[http://2009.igem.org/Team:Newcastle/Labwork/30_July_2009 30]	[http://2009.igem.org/Team:Newcastle/Labwork/31_July_2009 31]

August
M	T	W	T	F	S	S
					[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_August_2009&action=edit 1]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_August_2009&action=edit 2]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_August_2009&action=edit 3]	[http://2009.igem.org/Team:Newcastle/Labwork/4_August_2009 4]	[http://2009.igem.org/Team:Newcastle/Labwork/5_August_2009 5]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_August_2009&action=edit 6]	[http://2009.igem.org/Team:Newcastle/Labwork/7_August_2009 7]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_August_2009&action=edit 8]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_August_2009&action=edit 9]
[http://2009.igem.org/Team:Newcastle/Labwork/10_August_2009 10]	[http://2009.igem.org/Team:Newcastle/Labwork/11_August_2009 11]	[http://2009.igem.org/Team:Newcastle/Labwork/12_August_2009 12]	[http://2009.igem.org/Team:Newcastle/Labwork/13_August_2009 13]	[http://2009.igem.org/Team:Newcastle/Labwork/14_August_2009 14]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_August_2009&action=edit 15]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_August_2009&action=edit 16]
[http://2009.igem.org/Team:Newcastle/Labwork/17_August_2009 17]	[http://2009.igem.org/Team:Newcastle/Labwork/18_August_2009 18]	[http://2009.igem.org/Team:Newcastle/Labwork/19_August_2009 19]	[http://2009.igem.org/Team:Newcastle/Labwork/20_August_2009 20]	[http://2009.igem.org/Team:Newcastle/Labwork/21_August_2009 21]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_August_2009&action=edit 22]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_August_2009&action=edit 23]
[http://2009.igem.org/Team:Newcastle/Labwork/24_August_2009 24]	[http://2009.igem.org/Team:Newcastle/Labwork/25_August_2009 25]	[http://2009.igem.org/Team:Newcastle/Labwork/26_August_2009 26]	[http://2009.igem.org/Team:Newcastle/Labwork/27_August_2009 27]	[http://2009.igem.org/Team:Newcastle/Labwork/28_August_2009 28]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_August_2009&action=edit 29]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_August_2009&action=edit 30]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_August_2009&action=edit 31]

September
M	T	W	T	F	S	S
	[http://2009.igem.org/Team:Newcastle/Labwork/1_September_2009 1]	[http://2009.igem.org/Team:Newcastle/Labwork/2_September_2009 2]	[http://2009.igem.org/Team:Newcastle/Labwork/3_September_2009 3]	[http://2009.igem.org/Team:Newcastle/Labwork/4_September_2009 4]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_September_2009&action=edit 5]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_September_2009&action=edit 6]
[http://2009.igem.org/Team:Newcastle/Labwork/7_September_2009 7]	[http://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 8]	[http://2009.igem.org/Team:Newcastle/Labwork/9_September_2009 9]	[http://2009.igem.org/Team:Newcastle/Labwork/10_September_2009 10]	[http://2009.igem.org/Team:Newcastle/Labwork/11_September_2009 11]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_September_2009&action=edit 12]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_September_2009&action=edit 13]
[http://2009.igem.org/Team:Newcastle/Labwork/14_September_2009 14]	[http://2009.igem.org/Team:Newcastle/Labwork/15_September_2009 15]	[http://2009.igem.org/Team:Newcastle/Labwork/16_September_2009 16]	[http://2009.igem.org/Team:Newcastle/Labwork/17_September_2009 17]	[http://2009.igem.org/Team:Newcastle/Labwork/18_September_2009 18]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_September_2009&action=edit 19]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_September_2009&action=edit 20]
[http://2009.igem.org/Team:Newcastle/Labwork/21_September_2009 21]	[http://2009.igem.org/Team:Newcastle/Labwork/22_September_2009 22]	[http://2009.igem.org/Team:Newcastle/Labwork/23_September_2009 23]	[http://2009.igem.org/Team:Newcastle/Labwork/24_September_2009 24]	[http://2009.igem.org/Team:Newcastle/Labwork/25_September_2009 25]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_September_2009&action=edit 26]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_September_2009&action=edit 27]
[http://2009.igem.org/Team:Newcastle/Labwork/28_September_2009 28]	[http://2009.igem.org/Team:Newcastle/Labwork/29_September_2009 29]	[http://2009.igem.org/Team:Newcastle/Labwork/30_September_2009 30]

October
M	T	W	T	F	S	S
			[http://2009.igem.org/Team:Newcastle/Labwork/1_October_2009 1]	[http://2009.igem.org/Team:Newcastle/Labwork/2_October_2009 2]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_October_2009&action=edit 3]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_October_2009&action=edit 4]
[http://2009.igem.org/Team:Newcastle/Labwork/5_October_2009 5]	[http://2009.igem.org/Team:Newcastle/Labwork/6_October_2009 6]	[http://2009.igem.org/Team:Newcastle/Labwork/7_October_2009 7]	[http://2009.igem.org/Team:Newcastle/Labwork/8_October_2009 8]	[http://2009.igem.org/Team:Newcastle/Labwork/9_October_2009 9]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_October_2009&action=edit 10]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_October_2009&action=edit 11]
[http://2009.igem.org/Team:Newcastle/Labwork/12_October_2009 12]	[http://2009.igem.org/Team:Newcastle/Labwork/13_October_2009 13]	[http://2009.igem.org/Team:Newcastle/Labwork/14_October_2009 14]	[http://2009.igem.org/Team:Newcastle/Labwork/15_October_2009 15]	[http://2009.igem.org/Team:Newcastle/Labwork/16_October_2009 16]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_October_2009&action=edit 17]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_October_2009&action=edit 18]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_October_2009&action=edit 19]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_October_2009&action=edit 20]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_October_2009&action=edit 21]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_October_2009&action=edit 22]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_October_2009&action=edit 23]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/24_October_2009&action=edit 24]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_October_2009&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_October_2009&action=edit 26]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_October_2009&action=edit 27]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/28_October_2009&action=edit 28]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_October_2009&action=edit 29]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_October_2009&action=edit 30]	[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_October_2009&action=edit 31]

20 – 21 June 2009 - Europe workshop (London)
23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
23 October Practice Presentation (Newcastle)
23 October T-shirts are ready
27 October Practice Presentation (Sunderland)
27 October Poster is ready
30 October – 2 November 2009 - Jamboree (Boston)

Newcastle iGEM Twitter
[http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
[http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]

Team:Newcastle/Labwork/21 September 2009

From 2009.igem.org

Formal Lab Session - 21st September 2009

Stochastic switch team

Metal Sensing Team

Introduction

Practical Outline

Procedure

Analysing midi-prep digests

Double digesting pMUTIN4 midi-prep DNA

Cleaning up cotC PCR product

Excising digested pMUTIN4 from gel

Quantification of cotC and pMUTIN4

Chassis team

Introduction

Experiment procedure

PCR doulbe clone fragment

Midi Prep

Digest pMutin4 plasmid for ligation

Digest kinA fragment

Conclusion

Futher plan