Team:SDU-Denmark/labnotes8

From 2010.igem.org

(Difference between revisions)
(→Further Characterization of BBa_K274210 in MG1655 and TOP10)
(→Lab notes (8/30 - 9/5))
 
(5 intermediate revisions not shown)
Line 270: Line 270:
<br>
<br>
Results: Primers were confirmed working, the next step will be PFU pcr.<br>
Results: Primers were confirmed working, the next step will be PFU pcr.<br>
-
 
== Retinal ==
== Retinal ==
-
=== Coloni PCR on transformations of NinaB in PSB1C3, K343005 in PSB3C5 and K343005 in PSB1AK3 with the dobbelt terminator  ===
+
=== Gradient PCR on ninaB (after PCR with ninaB2fw and ninaB2rv) with ninaBfw and ninaBrv ===
-
Date: 30/09<br>
+
Date: 30/08<br>
-
Done by: Tommy<br>
+
Done by: Tommy & Marie<br>
Methods: PCR<br>
Methods: PCR<br>
-
Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
+
Protocols: CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]<br>
Notes: <br>
Notes: <br>
-
Because the first coloni PCR of the transformants didn't give us the the right pieces 32 new colonies where chosen:<br>
+
To finde the optimal anneling temperatur a gradient PCR from 60 to 75 degrees<br>
-
32-40: ninaB in PSB1C3<br>
+
"ninaB" was used as template and ninaB2fw and ninaB2rv was used as primers<br>
-
41-48: K343005 in PSB3C3<br>
+
-
49-64: k343005 in PSB1AK3<br>
+
-
 
+
-
VF2 and VR used as primers in the TAQ PCR:<br>
+
PCR Program:<br>
PCR Program:<br>
Line 309: Line 304:
<td style="vertical-align: top;">Annealing<br>
<td style="vertical-align: top;">Annealing<br>
</td>
</td>
-
<td style="vertical-align: top;">55 C<br>
+
<td style="vertical-align: top;">Grad. C<br>
</td>
</td>
<td style="vertical-align: top;">1 min<br>
<td style="vertical-align: top;">1 min<br>
Line 319: Line 314:
<td style="vertical-align: top;">72 C<br>
<td style="vertical-align: top;">72 C<br>
</td>
</td>
-
<td style="vertical-align: top;">2:30 min<br>
+
<td style="vertical-align: top;">4 min<br>
</td>
</td>
</tr>
</tr>
Line 347: Line 342:
</tr>
</tr>
</table>
</table>
-
Colonies 34, 37, 61 and 63 was selected for minipreps and freezing cultures.
+
Vary small amount of the product are observed, it is pooled and gel purifid, the nanodrop shows 372,3 and 377,1 ng/uL.
-
=== dobbelt transformation of K343005 in PSB1C3 and K274210 in PSB1A2 in to top10 and MG1655 E. Coli ===
+
=== Gel purification af ninaB from gradient PCR ===
-
Date: 1/10<br>
+
Date: 31/08<br>
Done by: Tommy & Marie<br>
Done by: Tommy & Marie<br>
-
Methods: transformation<br>
+
Methods: PCR<br>
-
Protocols: TR1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#TR1.1]<br>
+
Protocols: CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]<br>
Notes: <br>
Notes: <br>
-
Top10 and MG1655 E. coli competent cells where prepared according to protocol<br>
+
Gel purification was preformed on the pooled product from the gradient PCR according to the protocol<br>
-
Minipreps of K343005 and K274210 was used<br>
+
After the gel purification the 2 samples was pooled and nanodroped: 7.0 ng/ul<br>
-
1uL of K343005 and 3uL of K274210 was used becaus og their differens in concentration. The rest was done accordin to the transformations protocol.<br>
+
Furthere PCR was preformed on the purifid product<br>
-
=== Coloni PCR on dobbelt transformants of K343005 in PSB1C3 and K274210 in top10 and MG1655 E. coli ===
+
 
-
Date: 2/10<br>
+
=== Gel purification af ninaB from gradient PCR ===
-
Done by: Tommy<br>
+
Date: 1/09<br>
-
Methods: Coloni PCR<br>
+
Done by: Tommy & Marie<br>
 +
Methods: PCR<br>
Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
Notes: <br>
Notes: <br>
-
8 Colonies from each strain of E. Coli where chosen:<br>
+
PCR was run according to protocol to the following to this program<br>
-
1-8: K343005 in PSB1C3 and K274210 in PSB1A2 in top10<br>
+
Gel purified produckt was used as template and ninaBfw and ninaBrv was used as primers<br>
-
9-16: K343005 in PSB1C3 and K274210 in PSB1A2 in MG1655<br>
+
-
VF2 and VR used as primers in the TAQ PCR:<br>
+
-
 
+
PCR Program:<br>
PCR Program:<br>
<table style="text-align: left;" border="1" cellpadding="2"
<table style="text-align: left;" border="1" cellpadding="2"
Line 392: Line 385:
<td style="vertical-align: top;">Annealing<br>
<td style="vertical-align: top;">Annealing<br>
</td>
</td>
-
<td style="vertical-align: top;">55 C<br>
+
<td style="vertical-align: top;">Grad. C<br>
</td>
</td>
<td style="vertical-align: top;">1 min<br>
<td style="vertical-align: top;">1 min<br>
Line 402: Line 395:
<td style="vertical-align: top;">72 C<br>
<td style="vertical-align: top;">72 C<br>
</td>
</td>
-
<td style="vertical-align: top;">5 min<br>
+
<td style="vertical-align: top;">4 min<br>
</td>
</td>
</tr>
</tr>
Line 410: Line 403:
<td style="vertical-align: top;">rep<br>
<td style="vertical-align: top;">rep<br>
</td>
</td>
-
<td style="vertical-align: top;">29x<br>
+
<td style="vertical-align: top;">44x<br>
</td>
</td>
</tr>
</tr>
Line 430: Line 423:
</tr>
</tr>
</table>
</table>
-
Colonies 2, 7, 11 and 12 was selected for minipreps and freezing cultures.
 
-
=== Miniprep on ninaB in PSB1C3 and K343005 in PSB1AK3 ===
+
=== Restriction digest of ninaB ===
-
Date: 2/10<br>
+
Date: 2/09<br>
-
Done by: Tommy<br>
+
Done by: Tommy & Marie<br>
-
Methods: Miniprep<br>
+
Methods: restriction digest<br>
-
Protocols: MP1.2[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2]<br>
+
Protocols: RD1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1]<br>
Notes: <br>
Notes: <br>
-
Colonies 34 and 37 contain ninaB in PSB1C3<br>
+
EcoRI and PstI ristriction enzymes was used and the ristriction digest was preformed according to protocol<br>  
-
Colonies 61 and 63 contain K343005 in PSB1AK3<br>
+
No gel purification was preformed, purificatin was preformed directly from the restriction mixture<br>
-
The miniprep was done according to protocol<br>
+
Purification was deno using the GFX purification kit and proformed according to that protocol, the purifid product was nanodroped: 3,1 ng/uL
-
The minipreps was dryed down and sendt to seqvensing<br>
+
-
===Further Characterization of BBa_K274210 in MG1655 and TOP10===
+
=== "Cross-PCR" on ninaB ===
-
Date: 3/10<br>
+
Date: 2/09<br>
-
Done by: Tommy<br>
+
Done by: Tommy & Marie<br>
-
Methods: Miniprep<br>
+
Methods: PCR<br>
-
Protocols: MP1.2[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2]<br>
+
Protocols: CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1]<br>
Notes: <br>
Notes: <br>
-
Further characterization of the BioBrick K274210 has been performed in Top10 and MG1655 E. coli. The experimental data have shown somewhat similar results for Top10 and MG1655 E. coli.
+
Tubes A: ninaBfw and ninaB2rv<br>
-
The experiment was performed as follows:<br>  
+
Tubes B: ninaB2fw and ninaBrv<br>
-
Over night (ON) cultures were grown from 4 colonies, until the following OD’s were obtained:  
+
NinaB PCR product with ninaB2fw and rv was used as template, both the tubes was run on the same PCR program
 +
<table style="text-align: left;" border="1" cellpadding="2"
 +
cellspacing="2">
 +
<tr>
 +
<td style="vertical-align: top;">Start<br>
 +
</td>
 +
<td style="vertical-align: top;">94&nbsp; C<br>
 +
</td>
 +
<td style="vertical-align: top;">2 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Denaturing<br>
 +
</td>
 +
<td style="vertical-align: top;">94 C<br>
 +
</td>
 +
<td style="vertical-align: top;">1 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Annealing<br>
 +
</td>
 +
<td style="vertical-align: top;">68 C<br>
 +
</td>
 +
<td style="vertical-align: top;">1 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Elongation<br>
 +
</td>
 +
<td style="vertical-align: top;">72 C<br>
 +
</td>
 +
<td style="vertical-align: top;">4 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Goto2<br>
 +
</td>
 +
<td style="vertical-align: top;">rep<br>
 +
</td>
 +
<td style="vertical-align: top;">44x<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">End<br>
 +
</td>
 +
<td style="vertical-align: top;">72 C<br>
 +
</td>
 +
<td style="vertical-align: top;">5 min<br>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td style="vertical-align: top;">Hold<br>
 +
</td>
 +
<td style="vertical-align: top;">4 C<br>
 +
</td>
 +
<td style="vertical-align: top;"><br>
 +
</td>
 +
</tr>
 +
</table>
-
Top 10 - no insert              OD = 0,008 (100 x diluted)<br>
 
-
Top 10 - with K274210 insert OD = 0,011 (100 x diluted)<br>
 
-
MG1655 - no insert OD = 0,020 (100 x diluted)<br>
 
-
Mg1655 - with K274210 insert OD = 0,017 (100 x diluted)<br>
 
-
ON cultures were grown in 110 ml LB media. Colonies with K274210 insert were grown in LB media containing ampicillin.
+
=== Miniprep of J13002 ===
-
All ON cultures were grown for 20 hours at 37 °C.
+
Date: 3/09<br>
-
After 16 hours, 10 ml of the ON cultures were transferred into 110 ml LB media and grown for 4 hours to reach the exponential phase, where the following OD’s were obtained:
+
Done by: Tommy & Marie<br>
 +
Methods: Miniprep<br>
 +
Protocols: MP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.1]<br>
 +
Notes: <br>
 +
The minipreps was done according to protocol<br>
 +
Nanodrop:<br>
 +
Tube 1: 38,9 ng/ul, 2nd elution: 20,5 ng/ul <br>
 +
Tube 2: 36,9 ng/ul, 2nd elution: 18,7 ng/ul <br>
 +
Tube 3: 38,4 ng/ul, 2nd elution: 20,0 ng/ul <br>
 +
Tube 4: 32,6 ng/ul, 2nd elution: 16,4 ng/ul <br>
-
Top 10 - no insert OD = 0,049 (100 x diluted)<br>
+
The pooled samples was nanodroped: 20,6 ng/uL
-
Top 10 - with K274210 insert OD = 0,044 (100 x diluted)<br>
+
-
MG1655 - no insert OD = 0,007 (100 x diluted)<br>
+
-
Mg1655 - with K274210 insert OD = 0,009 (100 x diluted)<br>
+
-
 
+
-
Cultures with K274210 insert were grown in media containing ampicillin.
+
-
100 mL cell culture were centrifuged for 5 min at 14000 RPM. The supernatant was discarded and cells were resuspended in 5 mL acetone (99,9%), except the Top 10 E. coli with the K274210 insert, which was resuspended in 10 mL acetone (source of error). The resuspended cells were sonicated for 5 min. Samples were spun down, the supernatant was transferred to new tubes, and cell debris was discarded. A standard curve was made from pure beta-carotene.<br>
+
-
 
+
-
The samples at the OD’s seen above as well as solutions of pure beta-carotene with known concentrations were measured at a fixed wavelength of 456 nm.
+
-
Known concentrations and their absorbances:
+
-
<table style="text-align: left; width: 100%;" border="1"
+
-
cellpadding="2" cellspacing="2">
+
-
    <tr>
+
-
      <td>Concentration</td>
+
-
      <td>Absorbance</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>1 mM</td>
+
-
      <td>4,000</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>100 µM</td>
+
-
      <td>2,260</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>50 µM</td>
+
-
      <td>4,000</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>25 µM</td>
+
-
      <td>0,155</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>10 µM</td>
+
-
      <td>0,893</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>5 µM&nbsp;</td>
+
-
      <td>0,440</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>1 µM</td>
+
-
      <td>0,075</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>100 nM</td>
+
-
      <td>0,015</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>10 nM</td>
+
-
      <td>0,038</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>1 nM</td>
+
-
      <td>0,005</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>100 pM</td>
+
-
      <td>0,024</td>
+
-
    </tr>
+
-
</table>
+
-
The samples and their absorbances:<br>
+
-
<table style="text-align: left; width: 100%;" border="1"
+
-
cellpadding="2" cellspacing="2">
+
-
    <tr>
+
-
      <td></td>
+
-
      <td>Top 10 cells (Absorbance)</td>
+
-
      <td>MG1655 E. coli mutant (Absorbance)</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>Stationary phase control </td>
+
-
      <td>0,034</td>
+
-
      <td>0,024</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>Stationary phase with K274210 biobrick insert</td>
+
-
      <td>0,319</td>
+
-
      <td>1,549</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>Expotential phase control</td>
+
-
      <td>0,020</td>
+
-
      <td>0,024</td>
+
-
    </tr>
+
-
    <tr>
+
-
      <td>Expotential phase with K274210 biobrick insert</td>
+
-
      <td>0,034</td>
+
-
      <td>0,033</td>
+
-
    </tr>
+
-
</table>
+
-
UV-VIS absorbance spectra of the known solutions were obtained, as well as spectra of the samples at the ODs shown above. The spectra of Top 10 and MG1655 in the stationary phase as well as selected spectra of known solutions are shown below:<br>
+
-
[[Image:Team-SDU-denmarkBeta-carotene.jpg |400px]] <br>
+
-
These results were obtained from one experiment, which will be replicated later for more precision.<br>
+

Latest revision as of 19:21, 16 October 2010

Lab notes (8/30 - 9/5)

Contents


Flagella


Since the previous FlhDCmut was wrongly mutated due to incorrect mutation primers, we are now back to square one with new correct primers. The next weeks the flagella-group are working according to the following plan:
1) Miniprep of plasmids with "wrong" FlhDCmut

2) Two-step PCR to get mutatet FlhDC
3) Cut and Ligate into pSB1C3 and pSB1AK3 and transform into TOP10 cells
4) Send to sequencing
5) Characterize biobrick

Miniprep of "wrong" FlhDCmut


Done by: Louise
Date: September 3rd
Protocol: [MP1.2]
Notes:
No changes of protocol were made.
Results: Nanodrop after sample was dried down:
Sample 1: Concentration: 192ng/ul Purity: 1.96/1.90
Sample 2: Concentration: 200ng/ul Purity: 1.84/1.88

The samples were run on a 1.5% gel with a 10kb ladder. The bands are positioned between 1500bp and 1200bp. FlhDCmut in pSB1C3 is 1248bp.
Team SDU-Denmark Miniprep af flhDCmut i 1C3.jpg

Photosensor

PCR on pKJ606 with PSfw and VR primers

Date: 31/8
https://2010.igem.org/wiki/index.php?title=Team:SDU-Denmark/labnotes8&action=edit&section=3 Done by: LC
Methods: PCR
Protocols: CP1.3[1]
Notes:
Premix:
7,5 µl 10xTAQ Buffer
3 µl MgCl2
3 µl VF2
3 µl VR
1,5 µl dNTP
55,5 µl H2O
3 µl template (PS1 miniprep)
3/8 µl TAQ Polymerase

PCR Program:

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



Results: The bands that showed up were around 2500 BP as expected from the sequencing results. This means that the designed primers have a high possibility of working, so that they will be ordered.
Team-SDU-Denmark-PSfwVR.jpg

PCR on pKJ606 with PSfw and PSrv primers

Date: 02/09
Done by: LC
Methods: PCR
Protocols: CP1.3[2]
Notes:
Premix:
7,5 µl 10xTAQ Buffer
3 µl MgCl2
3 µl VF2
3 µl VR
1,5 µl dNTP
55,5 µl H2O
3 µl template (PS1 miniprep)
3/8 µl TAQ Polymerase

PCR Program:

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



Results: The bands that showed up were around 1750 BP, further confirming sequencing results. New primers for taking the whole gene out have been ordered.
Team-SDU-Denmark-PSfwrv.jpg

PCR on pKJ606 with fwPS2 and rvPS2 primers

Date: 04/09
Done by: LC
Methods: PCR
Protocols: CP1.3[3]
Notes:
Premix:
12,5 µl 10xTAQ Buffer
5 µl MgCl2
5 µl VF2
5 µl VR
2,5 µl dNTP
59,5 µl H2O
4 µl template (Consisting of 2 µl H2O and 2 µl of pKJ606 miniprep)
1/2 µl TAQ Polymerase

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
 3 min
Hold
 4 C



Results: Primers were confirmed working, the next step will be PFU pcr.

Retinal

Gradient PCR on ninaB (after PCR with ninaB2fw and ninaB2rv) with ninaBfw and ninaBrv

Date: 30/08
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.1[4]
Notes:
To finde the optimal anneling temperatur a gradient PCR from 60 to 75 degrees
"ninaB" was used as template and ninaB2fw and ninaB2rv was used as primers

PCR Program:

Start
 94  C
 2 min
Denaturing
 94 C
 1 min
Annealing
 Grad. C
 1 min
Elongation
 72 C
 4 min
Goto2
 rep
 29x
End
 72 C
 5 min
Hold
 4 C

Vary small amount of the product are observed, it is pooled and gel purifid, the nanodrop shows 372,3 and 377,1 ng/uL.

Gel purification af ninaB from gradient PCR

Date: 31/08
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.1[5]
Notes:
Gel purification was preformed on the pooled product from the gradient PCR according to the protocol
After the gel purification the 2 samples was pooled and nanodroped: 7.0 ng/ul
Furthere PCR was preformed on the purifid product


Gel purification af ninaB from gradient PCR

Date: 1/09
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.3[6]
Notes:
PCR was run according to protocol to the following to this program
Gel purified produckt was used as template and ninaBfw and ninaBrv was used as primers
PCR Program:

Start
 94  C
 2 min
Denaturing
 94 C
 1 min
Annealing
 Grad. C
 1 min
Elongation
 72 C
 4 min
Goto2
 rep
 44x
End
 72 C
 5 min
Hold
 4 C

Restriction digest of ninaB

Date: 2/09
Done by: Tommy & Marie
Methods: restriction digest
Protocols: RD1.1[7]
Notes:
EcoRI and PstI ristriction enzymes was used and the ristriction digest was preformed according to protocol
No gel purification was preformed, purificatin was preformed directly from the restriction mixture
Purification was deno using the GFX purification kit and proformed according to that protocol, the purifid product was nanodroped: 3,1 ng/uL

"Cross-PCR" on ninaB

Date: 2/09
Done by: Tommy & Marie
Methods: PCR
Protocols: CP1.1[8]
Notes:
Tubes A: ninaBfw and ninaB2rv
Tubes B: ninaB2fw and ninaBrv
NinaB PCR product with ninaB2fw and rv was used as template, both the tubes was run on the same PCR program

Start
 94  C
 2 min
Denaturing
 94 C
 1 min
Annealing
 68 C
 1 min
Elongation
 72 C
 4 min
Goto2
 rep
 44x
End
 72 C
 5 min
Hold
 4 C


Miniprep of J13002

Date: 3/09
Done by: Tommy & Marie
Methods: Miniprep
Protocols: MP1.1[9]
Notes:
The minipreps was done according to protocol
Nanodrop:
Tube 1: 38,9 ng/ul, 2nd elution: 20,5 ng/ul
Tube 2: 36,9 ng/ul, 2nd elution: 18,7 ng/ul
Tube 3: 38,4 ng/ul, 2nd elution: 20,0 ng/ul
Tube 4: 32,6 ng/ul, 2nd elution: 16,4 ng/ul

The pooled samples was nanodroped: 20,6 ng/uL

Retrieved from "http://2010.igem.org/Team:SDU-Denmark/labnotes8"