Back to iGEM 2010

Lab notes (8/2 - 8/8)

Contents 1 Lab notes (8/2 - 8/8) 1.1 Group: Flagella 1.1.1 Checking if Pst1 site in FlhDC mut is not pressent vol.2 1.1.1.1 vol. 3 1.1.1.2 Vol. 4 - Fourth time is the charme 1.1.2 Colony PCR of FlhDCmut and DT (B0015) 1.1.3 Colony PCR of pSB3T3 w RFP(J04450) 1.1.4 Colony PCR of pSB3C5 w RFP(J04450) 1.1.5 Insertion of B0015 in pSB3C5 1.1.5.1 pfu PCR of B0015 and PCR purification 1.1.5.2 Digestion of B0015 and pSB3C5 1.1.5.3 Ligation 1.1.5.4 Transformation 1.1.5.5 Coloni PCR 1.2 Group: Photosensor 1.2.1 Restriction digest of PUC19 and the photosensor in pKJ606 with Nde1 1.2.2 Dreamtaq Green PCR Master Mix (2X) PCR on photosensor with VF2 and VR 1.2.3 pfu PCR of photosensor

Group: Flagella

Checking if Pst1 site in FlhDC mut is not pressent vol.2

Done by: Sheila & Louise
Date: August 2nd
Protocol: RD1.1
Notes: Freeze tube 9 containing DNA purification from MG1655 cells was used as native FlhDC. Freeze tube 48 containing FlhDC mut purification. These two samples was amplified according to CP1.1 and run with an annealing temperature of 63 celcius and 2 minutes elongation.
The PCR samples were loaded on a1.5% gel with two ladders (100-1000 and 100-10000) FlhDC native was loaded in the first 5 lanes and FlhDC mut in the next 5 lanes.
The gel is expected to show two bands for the native FlhDC gene, one arround 900bp and one arround 100bp, while the FlhDC mut is expected to show only one band, between 900bp and 1000bp.
Results: The gel is pour, shows smear and bands at 200 bp.

We suspect that the primers are to blame for this result. It is possible that when the primer-stock used was made, old primers was used instead of the new longer ones. If this is the case then the annealing temperature of 63 celcius (ajusted to the new primers) is way higher than that of the old ones (45 celcius) which would make it impossible for primers to anneal.
So we made new primer-stock, and removed the old stock and primers.
--Louch07 08:20, 5 August 2010 (UTC)

vol. 3

Done by: Sheila & Louise
Date: August 3th
Protocol: RD1.1
Notes: The previous experiment was redone with the new primers.
Five samples of native FlhDC (FT 9) and 3 samples of FlhDC mut (FT 48) was loaded on a 1.5% gel with two ladders (100-1000 and 100-10000).
Results: The gel still shows smear and bands at 200bp.
We have no explanation this time, and have desided to shelve this experiment a while.

--Louch07 10:20, 5 August 2010 (UTC)

Vol. 4 - Fourth time is the charme

Done by: Pernille & Louise
Date: August 5th
Protocol: RD1.1
Notes:

Cutting FlhDC
tube 1: FlhDC uncut (freeze tube 5)
tube 2: FlhDC cut with pst1 (5)
tube 3: FlhDC uncut (freeze tube 21)
tube 4: FlhDC cut with pst1 (21)

Cutting of FlhDCmut in pSB3K3
tube 5: FlhDCmut uncut (PCR product F, from august 4th)
tube 6: FlhDCmut cut with pst1 (F)
tube 7: FlhDCmut cut with spe1 (F)

tube 8: FlhDCmut uncut (PCR product G, from august 4th)
tube 9: FlhDCmut cut with pst1 (G)
tube 10: FlhDCmut cut with spe1 (G)

Cutting of FlhDCmut in pSB1C3
tube 11: FlhDCmut uncut (PCR product C, from august 4th)
tube 12: FlhDCmut cut with pst1 (C)
tube 13: FlhDCmut cut with spe1 (C)

tube 14: FlhDCmut uncut (PCR product D, from august 4th)
tube 15: FlhDCmut cut with pst1 (D)
tube 16: FlhDCmut cut with spe1 (D)


Restriction mixture
13ul water
2ul FD green buffer
5ul PCR product
1ul enzyme

Results:
In the picture U = Uncut, P = Pst1 and S = Spe1. The Native FlhDC has an internal Pst1 site, while this site has been altered in the FlhDCmut gene. As expected all the cut samples show two bands, while the uncut samples only show one band. The picture shows a difference between the cut band and the greatest cut band in the native FlhDC, the cut band is slightly leighter than the uncut (ca. 100bp). The FlhDCmut samples show no specefic difference between the uncut band and the greatest cut band.
This indicates that a grater part of the native FlhDC is cut of than is cut of the FlhDCmut.

Colony PCR of FlhDCmut and DT (B0015)

Done by: Maria, Pernille & Louise
Date: August 4th
Protocol: CP1.3
Notes: The FlhDC mut gene was previously incearted into two different plasmids (pSB3k3 and pSB1c3) and the DT was incearted into pSB3c5. These were grown and 8 colonies from different plates were picked

FlhDCmut in pSB3k3, PCR-tube 1a-h

- 1a, -b, -c were picked from plate L1.1b (30.07.10) 

- 1d, -e, -f were picked from plate L2.1a (30.07.10) 

- 1g, -h were picked from plate L3.2a (30.07.10) 

FlhDCmut in pSB1c3, PCR-tube 2a-h

- 2a, -b were picked from plate L1.3b (30.07.10) 

- 2c, -d, -e were picked from plate L3.2b (30.07.10) 

- 2f, -g, -h were picked from plate L2.1b (30.07.10) 

DT(B0015) in pSB3c5, PCR-tube 3a-h*

- 3a, -b were picked from plate L1.2a (30.07.10) 

- 3c, -d were picked from plate L2.1c (30.07.10) 

- 3e, -f were picked from plate L2.1b (30.07.10) 

- 3g, -h were picked from plate L2.2a (30.07.10) 

̈́* On these plates were many red colonies, this might be because no extraction was done after the cutting, which would result in un-cut plasmids to be pressent.

Pre-mix x 25
87.5 ul water
62.5 ul 10 x TAQ buffer
25 ul MgCl2
25 ul VF2
25 ul VR
12.5 ul 10mM dNTP
3 ul TAQ (we've got a new TAQ stock which is twice as strong as the previous, so now we only use 1/8 TAQ of the amount in the protocol. ((1/8 x 1 ul) x 24))

PCR Program:

Progress	Temp (celcius)	Time (min) FlhDC	Time (min) DT
Start	94	2	2
Denaturing	94	1	1
Annealing	55	1	1
Elongation	72	1.5	0.5
GOTO 2
End	72	3	3
Hold	4

Results:
DT:
The double terminator was run on a 2% gel with a 100-1000 ladder. The gel shows no DNA at all!


FlhDCmut in pSB3k3:'
This was run on a 1% gel with a 50-2000bp ladder. The light in this picture is bad, but it does show high concentration of DNA in six of the eight ligations. The bands are positioned above 1000bp which is expected since the FlhDCmut gene with VF2-VR is 1248bp.


FlhDCmut in pSB1c3:
This was run on a 1% gel with a 50-2000bp ladder. The picture shows high concentration of DNA in seven of the eight ligations. The bands are positioned above 1000bp which is expected since the FlhDCmut gene with VF2-VR is 1248bp.

Colony PCR of pSB3T3 w RFP(J04450)

Done by: Pernille
Date: August 10th
Protocol: CP1.1
Notes: The experiment was done with Taq will the purpose only was to check if the RFP was amplificated when using the primers VR og VF2. The PRC program used was c.f. the protocol but the elongation time was 1min and 30 sec while the biobrick in the pSB3T3 plamid are 1319bp long. For the PCR i used the mini prep product from freeze tube 29.
Results:

Unfortunately there was no band around 1400bp and therefore the amplification was not succeed. the upper band is probably the plamid. --Pernm07 08:58, 11 August 2010 (UTC)

Colony PCR of pSB3C5 w RFP(J04450)

Done by: Pernille
Date: August 11th
Protocol: CP1.1
Notes: The experiment was done with Taq will the purpose only was to check if the RFP was amplificated when using the primers VR og VF2. The PRC program used was c.f. the protocol but the elongation time was 1min and 30 sec while the biobrick in the pSB3T3 plamid are 1319bp long. For the PCR i used the mini prep product from freeze tube 30.
Results:

Insertion of B0015 in pSB3C5

Done by: Maria and LC
Date: 4-5. august
Methods: PCR, PCR-purification, Digestion, Gel extraction, Ligation and transformation

pfu PCR of B0015 and PCR purification

date: 4/8 Protocols: CP1.1 and GFX easy protocol
Notes:
2 uL PCR product of B0015 (no. 43 white) was used as template for each PCR reaction.3 PCR reactions were prepared.
Premix for 4 PCR reactions:

20uL	pfu bf. + MgSO4
6uL	dNTP's
6uL	VF2
6uL	VR
152uL	H20
1.5uL	pfu Polymerase enzyme

48uL premix is distrubuted into each PCR tube. PCR tubes are marked B0015.A-C
PCR program:

Start	94C	3min
Denaturating	94C	2min
Annealing	55C	30s
Elongation	72C	45s
Go to	2	29x
End	72C	2min
Hold	4C

5uL of PCR sample is loaded onto a 2% agarose gel. Generuler 100bp DNA ladder (blue) is used as marker.

Results:


Analysis: PCR product is OK and B0015 DNA is purified from the PCR product according to protocol. DNA is eluted in 20uL H2O. Purified samples are pooled and used for digestion.

Digestion of B0015 and pSB3C5

date:4/8
Protocols:RD1.1DE1.3
Notes:
purified B0015 PCR product and mini prep of pSB3C5 (28 white) are digested with EcoRI and PstI.
Restriction mixture:

Restriction mixture B0015		Restriction mixture Plasmid
38uL	H2O	24uL
8uL	FD green buffer	4uL
4uL	EcoRI	2uL
4uL	PstI	2uL
30uL	DNA	10uL

Total volume of digested samples were loaded onto agarose gels and extracted from gel according to protocol. DNA was eluted in 20uL H2O.

Results:


Analysis:
Digested DNA was used for ligation.

Ligation

Date: 4/8
Protocol: LG1.2

Notes:
B0015 is ligated with pSB3C5.
3 ligation mixtures with ratios of 1:3, 1:6 and 1:9 (vector:B0015) was prepared.
45ng of vector was used for each ligation.
Ligation mixtures:

	Lig. 1(1:3)	Lig. 2 (1:6)	Lig. 3 (1:9)
T4 ligase bf.	2uL	2uL	2uL
T4 ligase	1uL	1uL	1uL
pSB3C5	3uL	3uL	3uL
B0015	2uL	4.5uL	6.5uL
H20	12uL	9.5uL	7.5uL

Ligations were incubated ON at 17degrees.

Transformation

Date: 5/8
Protocols: CC1.1TR1.1

Notes:
For compotent cells a Top 10 E.coli coloni was inoculated in 5mL LB media ON.
ON culture was diluted and grown to reach exponential phase.

Time	OD550
8:07	2.53
8:20	0.02
9:30	0.078
10:30	0.404

3 transformations was made for each of the ligation mixtures.

Results:
No colonies on the neg. controls
Many red colonies on the plate for the pos. control
Both red and white colonies were shown on the plates with the ligation mixtures.

Analysis:
The transformation appears to have been successful and white colonies where selected for coloni PCR.

Coloni PCR

Date: 5/8
Protocols: CP1.3

Notes:
8 white colonies were selected from L1, L2, and L3 plates repectively.
Premix x 10:

25uL	taq buffer (+KCl - MgCl2)
10uL	MgCl2
10uL	VF2
10uL	VR
5uL	d'NTP
35uL	H2O
1uL	taq polymerase enzyme

PCR tubes are marked Lig3.A-H
PCR program:

Start	94C	2min
Denaturating	94C	1min
Annealing	55C	1min
Elongation	72C	30s
Go to	2	rep. 29x
End	72C	3min
Hold	4C

PCR product was loaded onto a 2% agarose gel. Gene ruler 100bp DNA ladder (blue) was used as marker.

Results:

Analysis:
No visible results. Do-over!!

Group: Photosensor

Restriction digest of PUC19 and the photosensor in pKJ606 with Nde1

Start date: 2/08 End date: 2/08
Methods: Restriction digest, gel electrophoresis

Protocol:RD1.1

Experiment done by: LC

Notes: Since all PCR attempts with VF2 and VR had failed until now, we wanted to see if there was an insert at all in the physical DNA we received. Therefore we cut PUC19 and the photosensor to compare their lengths to each other, since the photosensor should be longer than PUC19 uncut and the cut photosensor should consist of two pieces, a 900 - 1000 BP piece and a 3600 BP piece.
Loading order:
1: PUC19 uncut
2: PUC19 cut
3: Photosensor uncut
4: Photosensor cut

Results:

Analysis:
We can clearly see that the photosensor uncut is longer than the uncut PUC19. We can also see multiple bands on the cut photosensor, which are the expected lengths. This indicates that the photosensor gene should be inserted between VF2 and VR, so we are still unsure why the PCR is not working.

Dreamtaq Green PCR Master Mix (2X) PCR on photosensor with VF2 and VR

Start date: 2/08 End date: 2/08
Methods: PCR, gel electrophoresis

Protocol: Dream TAQ Green PCR Master Mix protocol (modified)

Experiment done by: LC

Notes: Instead of a 50 µl reaction we ran a 25 µl reaction. It was mixed like this:

Dream TAQ Master Mix	12,5 µl
VF2	1 µl
VR	1 µl
Template	0,5 µl
Water	10 µl

We used a miniprep of the photosensor as template DNA, hence the small amount of template. The PCR program used was as follows:

Step	Temperature	Time	Number of cycles
Initial denaturation	95°	1 - 3 min	1
Denaturation	95°	1 min	30
Annealing	55°	1 min	30
Extension	72°	3 min	30
Final extension	72°	3 min	1

Results:

Analysis:
We got bands at 270 BP, 850 BP (the distance between VF2 and VR without insert) and two bands very high up in the gel (probably the template which did not get amplified and could not run through the 1,5% gel).

pfu PCR of photosensor

Date: 10/8
Experiment done by: Maria
Methods: PCR, gel electrophoresis
Notes:
Miniprep of photosensor in pJK606 was used as template. 2uL template was used for each reaction.
4 PCR reaction was prepared.
Premix x 5:

25uL	10 x pfu
7.5uL	VF2
7.5uL	VR
7.5uL	d'NTP
210uL	H2O
2uL	pfu polymerase enzyme

48uL premix is distributed into each PCR tube. PCR tubes are marked PS3.A-D.
PCR program:

Start	94C	3min
Denaturating	94C	2min
Annealing	55C	30s
Elongation	72C	4min 30s
Go to	2	rep. 29x
End	72C	2min
Hold	4C

PCR product are loaded onto a 1% gel and run at 130V. Gene ruler green was used as marker.

Results:

Analysis:
No usable results.

What up!