From 2010.igem.org

Lab notes (9/6 - 9/12)

Flagella Group

Two-step PCR of miniprep

First step: PCR of miniprep with mutation primers

Done by: Louise
Date: September 7th
Protocol: [CP1.1]
Notes:
3 x Premix 1:
114.3ul water
15ul Pfu Buffer
4.5ul dNTP
3ul MgSO4
4.5ul FlhDC fw
4.5ul FlhDCmut rev
1.2ul PFU
3ul template (miniprep)

3 x Premix 2:
114.3ul water
15ul Pfu Buffer
4.5ul dNTP
3ul MgSO4
4.5ul FlhDCmut fw
4.5ul FlhDC rev
1.2ul PFU
3ul template (miniprep)

Results:
NanoDrop:
Sample 1.1: Concentration: 359.5ng/ul Purity: 1.83/2.20
Sample 2.1: Concentration: 304.1ng/ul Purity: 1.85/2.23

The PCR samples were run on a 1.5% gel with a 100bp-1000bp ladder. The first three samples are all positioned between 100bp and 200bp, this is the part of the gene run with FlhDCmut fv and FlhDC rev primers. The other three samples are all positioned betveen 900bp and 1000bp, this is the pcr with FlhDC fv and FlhDCmut rev primers. All samples looked okay and were pooled as sample 1.1 (light band) and sample 2.1 (heavy band)

A gel extraction of the PCR product of sample 1.1 and 2.1 were made:

The gel extraction products were used in the preceding PCRs.

Second step: 2-step PCR with mutated template sample 1.1 and 2.1 and FlhDC fw and rev primers

Done by: Maria
Date: September 9th
Protocol: [CP1.1]
Notes:
Premix:
38ul water
5ul PFU buffer + MgSO4
1.5ul dNTP
1ul Sample 1.1
1ul Sample 2.1
1.5ul FlhDC fw primer
1.5ul FlhDC rev primer
0.5ul PFU

PCR Program:

1:Start	95C	2 min
2: Denaturing	95C	30 sec
3: Annealing	56C	30 sec
4: Elongation	72C	2 min
5:	GO TO	2 rep. 4x
6: Denaturing	95C	30 sec
7: Annealing	63C	30 sec
8: Elongation	72C	2 min
9:	GO TO	6 rep. 25x
10: End	72C	5 min
12: Hold	4C

Results:
Some of the PCR product was run on a 1.5% gel with a 10kb ladder.

The rest of the product was extracted from a new gel, unfortunately there were problems with the camera, so there is no picture of the gel extraction.

PCR of Gel extraction

Done by: Maria
Date: September 9th
Protocol: [CP1.1]
Notes:
Premix x 6:
234ul water
30ul PFU buffer + MgSO4
9ul dNTP
9ul FlhDC fw primer
9ul FlhDC rev primer
2.5ul PFU
6ul template

1:Start	95C	2 min
2: Denaturing	95C	30sec
3: Annealing	63C	30 sec
4: Elongation	72C	2 min
5:	GO TO	2 rep. 29x
6: End	72C	5 min
7: Hold	4C

Results:
The PCR worked fine, unfortunately there were problems with the camera, so there is no picture of the gel.

Restriction Digest of Gel Extracted FlhDCmut, pSB1C3 and pSB1AK3

Date: 9/12 2010
Done By: Sheila
Protocol: RD1.1
Notes: The restriction digests are made in preperation for two different ligations, the first is the ligation of FlhDCmut into the submission backbone; pSB1C3. The second is the the ligation of FlhDCmut into pSB1AK3. Different restriction enzymes were used in, as shown in the following tables

Restriction Digest mixtures
FlhDCmut and pSB1C3 Restriction digest
FlhDCmut

H2O	48μL
EcoR1	4μL
Pst1	4μL
FD Green buffer	8μL
Sample	20μL

pSB1C3

H2O	24μL
EcoR1	2μL
Pst1	2μL
FD Green buffer	4μL
Sample	10μL

FlhDCmut and pSB1AK3 Restriction digest
FlhDCmut

H2O	48μL
EcoR1	4μL
Spe1	4μL
FD Green buffer	8μL
Sample	20μL

pSB1AK3

H2O	24μL
EcoR1	2μL
Xba1	2μL
FD Green buffer	4μL
Sample	10μL

Following digestion, the samples were loaded and run on a 1.5% agarose gel, before being extracted prior to ligation.

Results:

Ligation of FlhDCmut and pSB1C3

Date: 9/12 2010
Done By: Sheila
Protocol: LG1.2
Notes:
Results:

Ligation of FlhDCmut and pSB1AK3

Date: 9/12 2010
Done By: Sheila
Protocol: LG1.2
Notes:
Results:

Colony PCR of FlhDCmut in pSB1AK3 and pSB1C3

Date: 9/15 2010
Done By: Louise
Protocol: CP1.3
Notes: We made 8 C-PCRs with colonies transformed with pSB1AK3 and 7 C-PCRs with colonies transformed with pSB1C3 (we made 8 but due to a mishab sample 5 was not run. Taq polymerase, VF2- and VR primers were used. Only Elongation time was altered from 2 min in the protocol to 1.5 min
Results:
The picture first picture is of FlhDCmut in pSB1AK3. This shows one band at about 400bp, the double terminator. This indicates that non of the colonies had the right plasmid.
The second picture is of FlhDCmut in pSB1C3.This shows the 4 of the 7 colonies had plasmids containing the right incert (sample 1,3, 7 and 8). These samples show a band at approximately 1200bp (FlhDCmut in pSB1C3 is 1248bp). the three other samples show a band heavier than 2000bp

Colony PCR of FlhDCmut in pSC1AK3

Date: 9/15 2010
Done By: Louise
Protocol: CP1.3
Notes: We made 8 C-PCRs with colonies transformed with pSB1AK3 and 7 C-PCRs with colonies transformed with pSB1C3 (we made 8 but due to a mishab sample 5 was not run. Taq polymerase, VF2- and VR primers were used. Only Elongation time was altered from 2 min in the protocol to 1.5 min
Results:

Photosensor group

Taq gradient PCR of pKJ606 with PS primers

Date: 9/7 2010
Done By: Maria and Lc
Protocol: CP1.3
Notes:
A gradient PCR was carried out to determine the appropiate annealing temperature of the new PS primers. PCR tubes were marked PSII A-H.

Premix x 9

Taq buffer (10x)	22.5uL
MgCl2	9uL
PSII fw primer	9uL
PSII rv primer	9uL
dNTP mix	4.5uL
H20	158.5uL
Taq polymerase	1uL

miniprep of pKJ606 was used as template. 1uL was distrubuted into each tube.
PCR program:

start	95C	2min
denaturating	95C	1min
annealing	se additional table	1min
elongation	72C	2min
go to	2	rep.29x
end	72C	5min
hold	4C

Anealing temperatures:

56.5C

58.3C

60.7C

63.3C

66C

68.6C

71C

73C

PCR product was loaded onto a 1.5 agarose gel. gene ruler DNA ladder mix (red) was used as marker.

Results:

Analysis:
Only very weak bands was observed at app. 2000 bp, and very strong bands were observed at app. 4000 bp, indicating that too much template was used in the PCR. An additional PCR was carried out using diluted miniprep as template.
--Tipi 10:27, 21 September 2010 (UTC)

Insertion of PS in pSB1C3 and pSB1AK3

Date: 9/8 - 9/12 2010
Done By: Maria and Lc
Protocol: CP1.1 DE1.3 RD1.1 LG1.2 CC1.1 TR1.1 CP1.3

Pfu PCR amplification of PS (no.1)

Date: 9/8 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
5 PCR reactions are prepared. 2uL Miniprep of pKJ606 are diluted in 8uL H2O to reach a 5x dilution, and are used as template. PCR tubes are marked PSIII A-E.
Premix x6:

pfu buffer + MgSO4	30uL
dNTP mix	9uL
PSII fw primer	9uL
PSII rv primer	9uL
H20	230uL
pfu polymerase	2.5uL
pKJ606 miniprep (5x diluted)	10uL

PCR program:

start	94C	3min
denaturating	94C	2min
annealing	68C	30s
elongation	72C	2min30s
go to	2	rep.29x
end	72C	5min
hold	4C

All of the PCR product was loaded onto a 1.5 agarose extraction gel. Gene ruler DNA ladder mix was used ad marker.

Results:
Analysis:
No bands were visible in the gel, indicating that the PCR reaction had been unsuccessfull. This could be due to the low elongation time, wherefore another PCR reaction with a longer elongation time was carried out.

Pfu PCR amplification of PS (no.2)

Date: 9/9 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
5 PCR reactions are prepared. 2uL Miniprep of pKJ606 are diluted in 8uL H2O to reach a 5x dilution, and are used as template. PCR tubes are marked PSIV A-E.
Premix x6:

pfu buffer + MgSO4	30uL
dNTP mix	9uL
PSII fw primer	9uL
PSII rv primer	9uL
H20	228uL
pfu polymerase	2.5uL
pKJ606 miniprep (5x diluted)	12uL

PCR program:

start	94C	3min
denaturating	94C	2min
annealing	68C	30s
elongation	72C	4min20s
go to	2	rep.29x
end	72C	5min
hold	4C

All of the PCR product was loaded onto a 1.5 agarose extraction gel. Gene ruler DNA ladder mix was used ad marker.

Results:
Analysis:
Bands were observed at app. 2000bp and bands were extracted by gel extraction

Gel extraction of PS

Date: 9/9 2010
Done By: Maria and Lc
Protocol:DE1.3
Notes:
DNA was extracted from gel according to protocol and each sample was diluted in 20uL.

Analysis:
each sample had a concentration of app. 7ng/uL. All 4 samples were pooled and used for restriction digest.

Restriction digest of PS, pSB1C3 and pSB1AK3

Date: 9/9 2010
Done By: Maria and Lc
Protocol:RD1.1 DE1.3
Notes:
Restriction mixture pSB1C3:

H2O	24uL
FD green buffer	4uL
EcoRI	2uL
PstI	2uL
pSB1C3	10uL

Restriction mixture pSB1AK3:

H2O	24uL
FD green buffer	4uL
EcoRI	2uL
XbaI	2uL
pSB1AK3	10uL

Restriction mixture PS (used in pSB1C3):

H2O	38uL
FD green buffer	8uL
EcoRI	4uL
PstI	4uL
PS	30uL

Restriction mixture PS (used in pSB1AK3):

H2O	38uL
FD green buffer	8uL
EcoRI	4uL
SpeI	4uL
PS	30uL

Digested samples were loaded onto a 1.5% agarose extraction gel. Uncut PS, pSB1C3 and pSB1AK3 were used as controles. Gene ruler DNA ladder mix was used as marker.
Loading scheme:

Lane	sample
1	marker
2	PS (used in pSB1C3)
3	uncut PS
4	PS (used in pSB1AK3)
5	uncut pSB1C3
6	pSB1C3
7	uncut pSB1AK3
8	pSB1AK3

DNA was extracted from gel according to protocol.

Results:
DNA conc:

sample	conc. (ng/uL)
PS (used in pSB1C3)	5.5
PS (used in pSB1AK3)	4.2
pSB1C3	6.3
pSB1AK3	14.3

Analysis: the purified DNA was used for ligation.

Ligation of PS and pSB1C3 and pCB1AK3

Date: 9/9 2010
Done By: Maria and Lc
Protocol:LG1.2
Notes:
For each of the ligations three ligation mixtures were prepared. vector concentrations of 10ng/uL (pSB1C3) and 15ng/uL (pSB1AK3) respectively was used for each mixture. Appropiate amount of insert was added to reac vector:insert ratios of 1:1, 1:2 and 1:4 respectively.
Ligation mixtures (PS in pSB1C3):

	L1	L2	L3
T4 ligase buffer	2uL	2uL	2uL
T4 ligase	1uL	1uL	1uL
pSB1C3	1.5uL	1.5uL	1.5uL
PS	1.5uL	3uL	6uL
H20	14uL	12.5uL	9.5uL

Ligation mixtures (PS in pSB1AK3):

	L1	L2	L3
T4 ligase buffer	2uL	2uL	2uL
T4 ligase	1uL	1uL	1uL
pSB1C3	1uL	1uL	1uL
PS	2uL	4uL	8uL
H20	14uL	12uL	8uL

The samples was incubated at 17C ON at used for transformation

Transfomation of ligated plasmin in Top 10 E.coli

Date: 9/10 2010
Done By: Maria and Lc
Protocol:CC1.1 TR1.1
Notes:
The compotent cells and transformation was carried out according to protocol. Cells transformed with ligations of PS in pSB1AK3 was plated on kanamycine plates.

Results:
the controle plates were okay, and there was many colonies on plates with cells transformed with ligations of pSB1C3 and PS. There was 10-20 colonies on the plates with cells transformed with ligation of PS and pSB1AK3.

Analysis:
The transformation was successfull and colonies was selected and used in coloni PCR.
--Tipi 13:41, 26 September 2010 (UTC)

Colony PCR of transformation of PS in pSB1C3 and pSB1AK3 in TOP10 E.Coli

Date: 09/11
Done by: LC & Maria
Methods: Colony PCR
Protocols: CP1.3[1]
Notes:
Premix:
45 µl 10xTAQ Buffer
18 µl MgCl2
18 µl VF2
18 µl VR
9 µl dNTP
63 µl H2O
2,25 µl TAQ Polymerase

The chosen colonies were lysed in 15 ul of H20.

PCR Program:

Start	94 C	2 min
Denaturing	94 C	1 min
Annealing	55 C	1 min
Elongation	72 C	2:30 min
Goto2	rep	29x
End	72 C	5 min
Hold	4 C

Results: For the ligation into pSB1C3, colonies B, F and H showed bands at the right length. For the other reatcion (in pSB1AK3), colonies A to D showed bands with the correct length. The colonies with bands at the right length will be miniprepped and sent for sequencing.

Control restriction digest of cPCR product of PS in pSB1C3 and pSB1AK3

Date: 09/12
Done by: LC & Maria
Methods: Restriction digest
Protocols: RD1.1[2]
Notes:
The afore mentioned samples (B, F and H of PS in pSB1C3 and A and D in pSB1AK3) were cut with EcoRI and PstI to see if it was 1) possible to cut them at all, thereby confirming the presence of biobrick prefix and suffix and 2) determining if the insert in the plasmid has the right length.
Mix for 1 RD reaction:
12 ul H2O
1 ul PstI
1 ul EcoRI
2 ul Fast digest green buffer
5 ul PCR product

Uncut PCR product and RFP were also loaded on the gel as a control.

Results: All samples pSB1C3 samples were cut as expected (to around 2000 BP), but the restriction digest of pSB1AK3 sample A and D failed, so that one will have to be repeated with sample B and C, as these only showed a cut double terminator without the PS insert.

Second control restriction digest of cPCR product of PS pSB1AK3

Date: 09/12
Done by: LC & Maria
Methods: Restriction digest
Protocols: RD1.1[3]
Since samples A and D fromt he colony PCR gave inadequate results when cut with EcoRI and PstI, we had to repeat the restriction digest with the remaining two samples that were the right length on the cPCR, sample B and C. Samples E, F and G were also cut to confirm that there were no errors in naming the samples that resulted in sample A and D not giving the expected results.
Restriction mixture:
72 ul H2O
6 ul PstI
6 ul EcoRI
12 ul Fast digest green buffer
5 ul PCR product per sample (x6)

Uncut PCR product was loaded on the gel as a control.

Results: Only sample C showed bands at the correct length (around 2180 BP), where as all other bands were at the length of a double terminator (b0015) without the photosensor insert. All samples were properly cut indicating the presence of biobrick pre- and suffix in these. Sample C will be miniprepped and sent off to sequencing.

Second round of colony PCR on PS ligated into pSB1AK3 (containing double terminator

Date: 09/12
Done by: LC & Maria
Methods: Colony PCR
Protocols: CP1.3[4]
Notes:
Since only sample C from the previous colony PCR looked right after a restriction digest we decided to run another round of colony PCR on the photosensor ligated into pSB1AK3, to find more colonys containing the right (ligated!) plamid.
The original samples A - D were also used as a template, now called I, J, K and L. Sample M to Y were new colonies.

Premix:
42,5 µl 10xTAQ Buffer
17 µl MgCl2
17 µl VF2
17 µl VR
8,5 µl dNTP
59,5 µl H2O
2,125 µl TAQ Polymerase

The chosen colonies were lysed in 15 ul of H20.

PCR Program:

Start	94 C	2 min
Denaturing	94 C	1 min
Annealing	55 C	1 min
Elongation	72 C	2:30 min
Goto2	rep	29x
End	72 C	5 min
Hold	4 C

Results: Samples K (former C) and Q produced bands at the right length of around 2150 BP, which eans that sample Q will also be miniprepped and send to sequencing together with the previously chosen sample C (now K).

Miniprep and controle digestion of PS in pSB1C3 and pSB1AK3

Date: 9/13 2010
Done By: Maria and Lc
Protocol:MP1.2 RD1.1
Notes:
Coloni B, F and H of PS in pSB1C3 and coloni C of PS + double terminator in pSB1AK3 was miniprep'ed according to prtocol. To ensure that the plasmids contains the insert as expected, the miniprep is cut with EcoRI and PstI.
the 2 minipreps of each coloni are diluted in 20uL and 50uL respectively.
Restriction mixture x4:

H2O	48uL
FD green buffer	8uL
EcoRI	4uL
PstI	4uL
Plasmid	4x5uL

The digested products are loaded onto a 1% agarose gel. 2.5uL of undigested plasmid are used as controle. Gene ruler DNA ladder mix are used as marker
Loading scheme:

Lane	Sample
1	marker
2	digested plasmid from col. B
3	undigested plasmid from col. B
4	digested plasmid from col. F
5	undigested plasmid from col. F
6	digested plasmid from col. H
7	undigested plasmid from col. H
8	digested plasmid from col. C
9	undigested plasmid from col. C
10	marker

concentrations of miniprep was measured on nanodrop.

Results:
nanodrop:

sample	conc. ng/uL	260/280	260/230
PS i pSB1C3 B1	232.86	1.91	2.17
PS i pSB1C3 B2	134.5	1.93	2.17
PS i pSB1C3 F1	229.32	1.9	2.18
PS i pSB1C3 F2	144.11	1.92	2.15
PS i pSB1C3 H1	190.88	1.91	2.2
PS i pSB1C3 H2	137.54	1.89	2.15
PS + double terminator in pSB1AK3 C1	227.6	1.91	2.2
PS + double terminator in pSB1AK3 C2	138.57	1.92	2.18

Analysis:
In the lanes containing the digested pSB1C3 plasmids bands at app. 2000bp are observed corresponding to PS. The lane containing digested pSB1AK3 plasmids shows a band at app. 2100bp corresponding to the size of PS and the double terminator. Miniprep of coloni B, F and C are sent to sequentation.
--Tipi 14:44, 26 September 2010 (UTC)

Miniprep of PS+B0015 sample Q and pSB3T5 containing J13002

Date: 27/7
Methods: ON, Miniprep
Protocol: MP1.1
Experiment done by: LC & Maria
Notes: PS+B0015 sample Q (1) was eluded in 20 ul to get a concentration over 200 ng/ul, so we would directly be able to send it in for sequencing. All other samples were eluded in 50 ul of elution buffer.

Results: The concentrations were as followed:
PS+B0015 (1): 180 ng/ul
PS+B0015 (2): 130 ng/ul
pSB3T5+J13002 (1 and 2): 50 ng/ul

Sample Q did not fulfill DNA technologies sample specifications for sequencing we waited with sending it in, since we were expecting the results from sample C which was sent in earlier. Since those returned positive, sample Q was never sent in, since we just proceeded with sample C.

Team:SDU-Denmark/labnotes9