Back to iGEM 2010

Lab notes (7/12 - 7/18)

Contents 1 Lab notes (7/12 - 7/18) 1.1 Group: Flagella 1.1.1 Extraction of psb3k3 plasmids with incerted RFP from E. coli MG1655 1.1.1.1 Exp. 1 1.1.1.2 Exp. 2 1.1.1.3 Exp. 3 1.1.2 Amplification of FlhDC, FlhD and FlhC genes 1.1.3 Amplification of FlhDC with Pfu 1.1.4 Amplification of FlhDC with pfx 1.1.5 Digestion of FlhD/C with pstI 1.1.5.1 Exp. 1 1.1.6 Amplification of FlhDC with Taq 1.1.7 Amplification of pSB3K3 with Taq 1.1.8 Genomic DNA purification 1.1.9 Genomic DNA purification 1.1.10 Amplification of FlhDC regulon from purified cromosomal DNA using TAQ 1.1.11 Checking the new primers 1.2 Group: Photosensor 1.3 Group: Retinal 1.3.1 Transformation of TOP10 e. coli with BBa_K274210 1.3.2 Transformation of BBa_R0011 and BBa_I0500 1.3.3 Mini-prep of pSB1A2 w. B0034, pSB1AK3 w. B0015 and pSB3K3 w. J04450(transformation from 08/07) 1.3.4 Digestion of pSB1A2 w. B0034, pSB1AK3 w. B0015 and pSB3K3 w. J04450 and pSB1A2 using pstI(miniprep from 12/07) 1.3.5 Transformation of MG1655 e. coli with BBa_274210, BBa_E0040 and BBa_I0500 1.3.6 Miniprep of BBa_K098995 in pSB1A2 1.3.7 Extraction of BBa_B0034 from pSB1A2 plasmid using PFU 1.3.7.1 Preliminary PCR to amplify VF2-VR piece containing part and restriction sites 1.3.7.2 Further amplification of PCR product 1.3.7.3 Gel Extraction of PCR product 1.3.8 Miniprep and transformation of BBa_K274210 in pSB1A2 1.3.9 Coloni PCR of pSB1A2 w. E0040 (transformation 13/7)

Group: Flagella

Extraction of psb3k3 plasmids with incerted RFP from E. coli MG1655

Exp. 1

Methods: Miniprep, NanoDrop and gel electrophoresis

protocols: MP1.1

Notes: We used 9 ml ON culture (Lag phase cells), loaded 2ul sample and 8ul agarose loading dye on a 1,5% gel,used a DNA ladder mix (100-10000 nucleotides) as marker

Results: Nanodrop: Tube 1: 30.7 mg/ul Tube 2: 27.4 mg/ul

Gel electrophoresis: No result

--Louise 15:00, 9 July 2010 (UTC)

Exp. 2

Methods: gel electrophoresis

Notes: We ran another gel electrophoresis on the miniPrep sample form above. But now we loaded 4ul sample and 4ul agarose loading dye on a 1,5% gel,used a DNA ladder mix (100-10000 nucleotides) as marker.

Results: Gel electrophoresis: Bands were detected. The psb3k3 plasmid is 2750bp long and the RFP with generator is 1096 bp, which gives a band at about 4000bp compaired with the marker.

Exp. 3

Methods: MiniPrep, NanoDrop and gel electrophoresis.

protocols: MP1.1

Notes: We used 10 ml af a culture in Log phase (1ml cells from an ON culture + 9ml LB medium, incubated at 37 degrees celcius for 4 hours), loaded 4ul sample and 4ul agarose loading dye on a 1,5% gel,used a DNA ladder mix (100-10000 nucleotides) as marker.

Results: Nanodrop: Tube 1: 38.7 mg/ul Tube 2: 32.4 mg/ul.

Gel electrophoresis: Bands were detected.

--Louise 17:21, 12 July 2010 (UTC)

Amplification of FlhDC, FlhD and FlhC genes

Date: july the 9th

Experiment done by: Sheila and Pernille

Methods: PCR and Gel electrophoresis.

Protocols: CHP1.2

Notes: We decided to test our primers on previously purified cromosomal DNA. Examination of the primers showed that the FlhC reverse primer had a melting temperature of only 45˚C. Therefore we decided to run the samples on a gradient PCR. Simultaneously, we prepared 2 extra samples, isolating FlhD and FlhC, respectively. We did this because we wanted to see if our problems were caused because the combined gene-sequence was to long (932bp).
Because we just wanted to test our primers in this PCR, we used Taq polymerase, because although it doesn’t proofread, it is remarkably cheaper than Pfu. On the [http://www.fermentas.com/en/products/all/pcr-qpcr-rt-pcr/standard-pcr/ep028-taq-dna-native Fermentas homepage] we found that the annealing temperature for Taq is Tm-5 , which in this case means 40˚C. However, Taq polymerase is not very effective at temperatures under 50˚C so we designed the gradient to lies between 40 and 55˚C. The PCR machine chose the following temperatures:

Colum	Temperature (Celcius)
1	39.9
2	40.3
3	41.2
4	42.6
5	44.3
6	46.3
7	48.3
8	50.5
9	52.1
10	53.6
11	54.6
12	55.1

We then chose six temperatures to test:

Sample	Temperature (Celcius)
1	40.3
2	42.6
3	44.3
4	46.3
5	48.3
6	50.3

The following table shows the Gradient PCR program:

Temperature (Celcius)	Time (min)	Description
94	3	Initialization
94	2	Denaturation
40-55	½	Annealing
72	2 x 29	Elongation
72	5	Final Elongation
4		Hold

Results: The experiment was succesfull! We could detect FlhDC DNA at temperatures between 42.6˚C and 48.3˚C. FlhD DNA at temperatures between 40.3˚C and 44.3˚C and also between 48.3˚C and 50.3˚C. FlhC DNA at temperatures run between 40.3˚C and 50.3˚C.

--Sheila 08:46, 14 July 2010 (UTC) --Pernm07 09:41, 14 July 2010 (UTC)

Amplification of FlhDC with Pfu

Date: july 12th

Experiment done by: Pernille

methods: PCR and gel electophoresis

Protocols: CHP1.2

Notes: The purified chromosomal DNA was tested with Pfu; a polymerase with proofreading ability. The DNA was from the E. coil strain MG 1655 (tube 17) The annealing temperature was used fore the PCR was 44˚C. We decide use this annealing temperature while it is in the middle of the temperature span we successful used in the last experiment. Further we increas the elongation time to 1 min and 30 sec. Other than these changes we followed the PRC program in the protocol. the PCR product was run on a 1,5% agarose gel.

results: Unfortunately the experiment did not give any results on the gel.

--Pernm07 10:56, 13 July 2010 (UTC)
--Sheila 08:47, 14 July 2010 (UTC)

Amplification of FlhDC with pfx

Date: july 13th

Experiment done by: Ann and Pernille

methods: The experiment was perform in the E.coli strain, MG1655. The DNA purification was done in duplicates and are saved in the freezer in tube 8 and 9. (green label). There DNA concentration was measure by Nano Drop and there contantretion are 16,87ng/uL and 19,25ng/uL, respectively.

protocol: GP1.1 and CP1.1.

Notes: The experiment was generally preformed corresponding to the protocol but small modification was made. In the DNA purification experiment we used 300uL instead of 200uL of the overnight culture and it is important to remember that the 800uL precipitation sulution, containing 80uL solution and 720uL deionized water, are made directly before use.
because our PRC for the time being havent been successful we are trying to do PCR with pfx wich are another polymerase with proofreading ability. The PRC program used for running PRC with pfx are as described below:

1	start	94°C	2 min
2	Denaturing	94°C	1min
3	Annealing	48°C	1 min
4	Elongation	68°C	2 min
5	GOTO 2		Rep x 29
6	End	68°C	3min
7	Hold	4°C

results: there did not appear any lanes when we runed the gel electophoresis. Therefore the next step is to run PCR with Taq polymerase to check if the problem are the pfu polymerase or if the DNA is not purify properly.

--Pernm07 10:19, 13 July 2010 (UTC)
--Sheila 08:49, 14 July 2010 (UTC)

Digestion of FlhD/C with pstI

Exp. 1

Methods: digestion and gel electrophoresis

protocols: RD1.1

Notes:
To verify the restriction site at 822bp in the FlhD/C gene, FlhD/C PCR product from Taq PCR was digested with pstI restrction enzyme.
smaples were loaded onto a 2% agarose gel. Blue gene ruler were used as marker. undigested FlhD/C PCR product was used as controle.

Results:
Analysis:
lane 2 and 5 containing the samples with pstI shows a small DNA fragment of app. 100 bp. This verifies the presence of a pstI restriction site in the FlhD/C gene. In order to use this gene as a biobrick we need to insert a silent mutation.

Amplification of FlhDC with Taq

Date: july 14th

Experiment done by: Pernille

Methods: The experiment was performed in the E.coli strain, MG1655, from freezer tube 8 and 9 wich was also used for amplification of FlhDC with pfx. In this experiment PCR was runned with Taq polymerase. The experiment was preformed according to the CP1.1 protocol.
Protocol: CP1.1.

Results: we did not get any results

--Pernm07 07:51, 14 July 2010 (UTC)
--Sheila 09:08, 14 July 2010 (UTC)

---

Amplification of pSB3K3 with Taq

Start date: July 14th 2010 End Date: July 14th 2010

Methods: The purified pSB3K3 plasmids were amplified by PCR using Taq, as a test, before using the more expensive Pfu.

Protocol: CP1.2

Results:

Notes: The plasmids were collected from tubes green 10 and green 11 in the freezer. VR and VF2 primers were used.
The experiment were performed simultaneously with the Taq control version of the extraction of BBa_B0034 from pSB1A2 plasmid done by the phototaxis goup

--Sheila 09:08, 14 July 2010 (UTC)

Genomic DNA purification

Date: July 14th

Experiment done by: Pernille and Louise

Methods: We used a ON culture of MG1655 cells

Protocols: GP1.1

Notes: We made four tupes with 300ul MG1655 cell media instead of 200ul. In tupe 1 and 2 I unfortunately added 800ul un-diluted precipitation solution, in tupe 3 and 4 I diluted the precipitation solution according to the GP1.1 protocol.

Results: The concentration of the purified DNA was measured by Nano Drop. Tybe 1 and 2 did not contain any DNA wich we had expected because of the high concentration of precipitation soultion. The DNA concentration i tube 3 and 4 are 28,38ng/uL and 19,4ng/uL, respectively. Tube 3 and 4 are efterwards pooled and saved in the freezer (labeled with white tag 1) The purified DNA was run on a agarose gel to check if it actual contains DNA.

--Louise 10:44, 14 July 2010 (UTC) --Pernm07 09:33, 14 July 2010 (UTC)

Genomic DNA purification

Date: July 15th

Experiment done by: Sheila

Methods: The DNA purification was performed on an ON culure of the MG1655 E. coli strain. Three samples of 400µL culture were extracted, centrifuged, the supernatant removed and the pellet was redissolved in 200µL water

Protocol: GP1.1

Notes:

Results: Not a lot of DNA was detected in the NanoDrop spectrophotometer measurements. The samples yielded concentrations of 9,88ng/µL, 13,5ng/µL and 5,3ng/µL respectively. The gel showed 4 bands of DNA in each sample. The samples were located at 200bp, 950bp, 1500bp and >10000bp. The bands less visible in lane 3, which were loaded with sample 3, which also yielded the least concentration.

--Sheila 09:20, 15 July 2010 (UTC)

Amplification of FlhDC regulon from purified cromosomal DNA using TAQ

Experiment done by: Christian

Date: 14/7

Methods: PCR on purified cromosomal DNA

Protocols: CP1.2

Notes: 4 tubes loaded with DNA from freezer: 2x(8 green) and 2x(9 green). Both tubes were discarded as they were empty.
(8 Green) was dilluted 4x to make pipetting possible.

Primers used were FlhD fw and FlhC rw.

Anealing temperature was set to 48°C and elongation was set to 2 minutes.

1,5% Gel was loaded with red 10kb ladder.

Results:Band showed at around 3kb in both wells for (8 green) and one well for (9 green)

Analysis: The FlhDC regulon was not extracted.

--CKurtzhals 16:11, 15 July 2010 (UTC)

Checking the new primers

Experiment done by: Pernille and Louise

Date: July 15th and 16th

Protocols: CP1.2

Note: Polymerase used: TAQ

Methods: PCR on purified MG1655 chromosomal DNA and Gel electrophoresis.

We used the four new primers we designed and ordered: FlhDC fw, FlhDC rev, FlhD mut fw and FlhD mut rev.
We ran a gradient PCR with six different annealing temperatures for three primer combinations (Tables below).

PCR-mixes:

PCR-mix 1	PCR-mix 2	PCR-mix 3
36 ul Water	36 ul Water	36 ul Water
5 ul 10x TAQ Buffer	5 ul 10x TAQ Buffer	5 ul 10x TAQ Buffer
1 ul 10 mM dNTP	1 ul 10 mM dNTP	1 ul 10 mM dNTP
2 ul MgCl2	2 ul MgCl2	2 ul MgCl2
2 ul FlhDC fw Primer (Freeze tupe white 5)	2 ul FlhD mut fw Primer (Freeze tupe white 7)	2 ul FlhDC fw Primer (Freeze tupe white 5)
2 ul FlhD mut rev Primer (Freeze tupe white 6)	2 ul FlhDC rev Primer (freeze tupe white 4)	2 ul FlhDC rev Primer (Freeze tupe white 4)
1 ul Purified chromosomal DNA from MG1655 (Freeze tupe white 9)	1 ul Purified chromosomal DNA from MG1655 (Freeze tupe white 9)	1 ul Purified chromosomal DNA from MG1655 (Freeze tupe white 9)
1 ul TAQ (added just before PCR start)	1 ul TAQ (added just before PCR start)	1 ul TAQ (added just before PCR start)

PCR Program:

Time:	Temperature (Celcius):	Process:
3 min	94	Start
2 min	94	Denaturation
30 sec	Gradient	Annealing
2 min	72	Elongation
5 min	73	End
DONE!	4	Hold

Annealing Gradient:

Colum	Temperature (Celcius)
4	48.5
5	50.8
6	53.4
7	56.1
9	60.1
11	64.5

Gel electrophoresis
Method: We used a 1.5% gel and two Ladders (GenRuler)

Loading Table

Well No.	PCR Loading	Primers
1	GenRuler 100-10.000	-
2	3.4 (PCR-mix 3. Colum 4)	FlhDC fw + FlhDC rev
3	3.5	----II----
4	3.6	----II----
5	3.7	----II----
6	3.9	----II----
7	3.11	----II----
8	2.4	FlhD mut fw + FlgDC rev
9	2.5	----II----
10	2.6	----II----
11	2.7	----II----
12	2.9	----II----
13	2.11	----II----
14	1.4	FlhDC fw + FlhD mut rev
15	1.5	----II----
16	1.6	----II----
17	1.7	----II----
18	1.9	----II----
19	1.11	----II----
20	GenRuler 100-1000	-

Group: Photosensor

Group: Retinal

Transformation of TOP10 e. coli with BBa_K274210

Experiment continued from last week.

Colony PCR of K274210 in pSB1A2

Start date: July 12th End date: july 12th

Experiment done by: Christian, Lars Christian

Methods: Colony PCR (modified)

Protocol: CP1.2

Notes: MgCl2 was added as a gradient,sample 1-2 contain 2 ul (0,04255), sample 3-5 2,25ul (0,04787). We mistakenly added 30ul (instead of 17ul) premix to sample 6 - 9, so the concentration of MgCl2 is reduced. Sample 6 contains 2,25ul (0,0375), Sample 7-8 2,5ul (0,04167), Sample 9 2,75ul (0,04583).

Gel was loaded with DNA-ladder plus, with the upper marker at 5000bp.

Results: We found plasmids at the expected 5kbp marker in colonies 1-8. Successful plates were kept in incubator over night. They will be made into ON cultures and thereafter frozen.

Analysis: It seems we have transformed our cells with BBa_K274210. Confirmation will come when we run experiments to demonstrate presence of Beta-carotene.

Transformation of BBa_R0011 and BBa_I0500

Start date: 12/07 End date:14/07
Methods: ON culture, competent cells, transformation.
Protocols:CC1.1, TR1.1.

Making competent E.Coli TOP 10
Experiment done by: Christian, Maria and LC
Date: 12/07
Notes: Everything was done after protocol.
Results: We made competent cells for use within 48 hours.
Analysis:


Transformation of pBad and lacl promoter
Experiment done by: Christian, Maria and LC
Date: 12/07 - 13/07
Notes: Added 200ul of the un-pelleted cells, instead of 150ul to the agar plates.
Some of the agar dishes were damaged during drying and plating. We tried to use them anyways.

Results: No plates showed colonies, apart from the positive control
Analysis: Either our cells weren't competent, or our agar dishes might have been the cause.

Mini-prep of pSB1A2 w. B0034, pSB1AK3 w. B0015 and pSB3K3 w. J04450(transformation from 08/07)

Start date: 12/07 End date: 12/07
Methods: mini-prep

Protocol: MP1.2

Notes: samples were loaded into a 1% gel with a gene ruler red marker.

Results:

Analysis:
A clear band was seen in all lanes, so we have succesfully isolated DNA.
All bands appeared to be about 1000 kb smaller than the expected size. This could be due to the supercoiling of the plasmids. To verify this, the plasmids were cut with pstI Digestion of plasmids

Digestion of pSB1A2 w. B0034, pSB1AK3 w. B0015 and pSB3K3 w. J04450 and pSB1A2 using pstI(miniprep from 12/07)

Start date: 13/07 End date: 13/07
Methods: Digestion
Protocol: RD1.1
Experiment done by: Maria and Sheila

Notes:Digestion of the plasmids with a single restriction enzyme was done to ensure that the plasmids extracted from mini-prep has the correct size. Digestion was done with half the amount of restriction mixture

samples were loaded into a 1.5% gel with a gene ruler red marker

Results:

Analysis:
All bands had the size we expected for the plasmids. We therefore expect that we have isolated the right plasmids
The samples from miniprep 12/7 was pooled and stored as no. 24, 25, 26 and 27 at -20 degrees C

--Maria 09:37, 14 July 2010 (UTC)

Transformation of MG1655 e. coli with BBa_274210, BBa_E0040 and BBa_I0500

Start Date: 13/07 End Date:
Methods: ON cultures, Competent Cells, Transformation of competent cells.

Protocols: CC1.1, TR1.1

Experiment: Making Competent e. coli MG1655
Experiment done by: Christian
Date: 13/07

*ON cultures of strain MG1655 were put over the night before.
Notes: Eksponential growth was started at 09.30 with an OD of 0.19A. The cells were harvested at 10.26. OD was .048 at 10x dillution.

Cells were put in fridge at 11.00. They will be ready at 11.30 and good for 48 hours.

Results: Competent cells were made according to protocol. Apparently MG1655 strain reaches OD 0.5 at least an hour faster than TOP 10.

Analysis:It is important to measurement at 10 minute intervals after OD 0.20 when growing MG1655 strain.

Experiment: Transforming MG1655 strain with BBa_E0040 (GFP coding sequence) and BBa_I0500 (pBad arabinose inducible promoter)
Date: 13/07-2010

Notes: I0500 is located in pSB2K3. E0040 is in pSB1A2. 200ul cells were added to each tube. Positive control is RFP generator as usual. Negative control is just cells.

Only 1ul DNA material was transferred per tube, since it was taken from distribution plates.

Plates were dried at 42°C for 15 minutes, just prior to plating. Upon plating we discovered a problem with many of the ampicilin plates we made a couple of days ago. The agar breaks on the slightest contact, and are therfore impossible to use.

As a result of the defective agar plates, most of our cells carrying BBa_E0040 were ruined. We managed to save and plate out two batches, one from each tube, so we are hoping for the best tomorrow.

Results: Only cells with BBa_E0040 grew colonies overnight.

Miniprep of BBa_K098995 in pSB1A2

Start date: 13/07 End date: 13/07
Methods: Fermentas GeneJET plasmid miniprep kit

Protocol: MP1.1

Experiment done by: LC

Notes: Centrifuged the ON for 15 mins. at 3000 rpm. Loaded 10 ml of culture in each tube and added 500ul resuspension solution, then split it into two samples.

Results:
Results were as expected (correct), the plasmid was around 3000 bp long on the gel.(Insert Gel picture and nanodrop results table)

Analysis:All four samples turned out fine, so they were pooled and frozen. (for details see results)
Nr. 28 in the freezer = BBa_K098995

Extraction of BBa_B0034 from pSB1A2 plasmid using PFU

Preliminary PCR to amplify VF2-VR piece containing part and restriction sites

Date: 14/7
Methods: PCR of DNA in solutions
Protocol: CP1.1-PFU protocol

Experiment done by: Christian

Notes: DNA material from tube 27 was taken from the freezer. Primers were standard vr-vf2, also from freezer. PFU buffer with MgSO4 was used. No further MgSO4 was added. A TAQ control was done in a parallel experiment.

Protocol was followed to point, with the exeption of 1ul additional H2O being added because of an error. It will yield 51ul.

Program was run according to protocol. elongation time was set to 1 minute and 30 seconds.

A control on the same DNA was run using TAQ polymerase. It was run on the same protocol and program as a parallel experiment in the flagella group: Amplification of pSB3K3 with taq

Results: Bands showed in all four wells on the gel, located at the expected length of 250BP. Nanodrop showed some contamination, but good DNA concentration at around 345ng/ul.

Analysis: The PCR product might be to contaminated still, and there#s too little anyways. Therefore The PCR product will need to be further amplified.

Further amplification of PCR product

Date: 14/7
Methods: PCR of DNA in solutions
Protocol: CP1.1-PFU protocol

Experiment done by: Christian

Notes: 4 tubes were loaded, using one ul of DNA template from the above experiment. Protocol was followed to point.

The same PCR program was used (PF1 on machine 2), elongation time once again set to 1 minute and 30 seconds.

Remaining PCR product was frozen as (White-2).

Gel was loaded with 5ul samples of each tube and a blue ladder marker.

Results: Bands showed at 250BP for all tubes. Tubes 1-3 showed a strange band that was larger than the blue ladder could show. Tube 4 was free of this band, and also showed a clearer band at 250BP.

Nanodrop showed signatures for tubes 1-3 like in the samples of the above experiment. DNA was twice as concentrated in tube 4.

--CKurtzhals 16:05, 14 July 2010 (UTC)

Gel Extraction of PCR product

Date: 15/7
Methods: Gel Extraction Kit
Protocol: DE1.1
Experiment done by: Christian, Lars Christian

Notes: four 50ul wells were created in the gells, to accomodate all the genetic material, which was loaded along with a blue 1kb ladeder. We extracted DNA at 250BP position according to protocol.

after melting gel down, we added 100% isopropanol to solution in 1:2 ratio, as noted in the kit protocol for DNA pieces less than 500BP.

Lars Christian eluded twice with 30ul elution buffer instead of once with 50ul for 4 of the eight tubes. Otherwise protcol was followed.

Extractions were pooled and frozen as (White 11)

Results: Nanodrop showed 5,00ng/ul DNA with 260/280 ratio at 2.29 and 260/230 ratio at 0.02.

Analysis: This product is not optimal. We will need to decide wether to try again.

--CKurtzhals 16:07, 15 July 2010 (UTC)

Miniprep and transformation of BBa_K274210 in pSB1A2

Start date: 14/07 End date:
Methods: Fermentas GeneJET plasmid miniprep kit

Protocol: MP1.1

Experiment done by: LC

Notes: No pellet after step 4, but still continued. Later we got a small pellet at step 6.

Results:
The miniprep failed as expected, since there was no pellet after the second centrifugation.(insert gel picture)

Analysis:The experiment will be repeated in the afternoon.

Second try:

Methods and protocol as in the first try.
Notes: We only had 3,5 ml of ON culture for the miniprep, so we had to manage with that.
Results: Insert gel picture and nanodrop table.
Analysis: The second try worked, so we went on to make transformations with the plasmid. It is in the freezer as nr. 3 (white).

Transformation of the miniprepped plasmid:

Experiment done by: LC, Maria

Protocol: TR 1.1

Notes: TRansformed BBa_K274210 in pSB1A2 on LA Amp plates into Mg1655. We used 2 ul from the miniprep product for each sample.
Our suspicion that the agar plates we made are faulty got confirmed, three more plates were destroyed while spreading the bacteria on them (Sample 1a, 2b, 2pellet and the positive control)

Results: Cells grew just fine and the negative control was as expected. After a night at 37° the cells seemed a little more yellowish tot he eye, than the usual.

Analysis: The transformations succeeded.

Coloni PCR of pSB1A2 w. E0040 (transformation 13/7)

Start date: 14/07 End date:
Methods: taq coloni PCR and gel electrophoresis

Protocol: CP1.2

Experiment done by: Maria

Notes:10 colonies from the Transformation 13/7 were used.
In PCR tube no. 6 most of the premix was lost.

Results:


Analysis:
--Maria 10:19, 14 July 2010 (UTC)

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