Difference between revisions of "Team:Marburg/Labbook/InterLab"

Line 1,974: Line 1,974:
 
<h1>15/08/29</h1>
 
<h1>15/08/29</h1>
 
<p>
 
<p>
−
<h2>PCR of parts for pILS7-9</h2>
+
<h2>PCR </h2>
−
lalala<br>
+
Amplify parts for pILS7-9<br>
 
<br>
 
<br>
  
Line 1,990: Line 1,990:
 
<br>
 
<br>
  
 +
 +
 +
<h1>15/08/30</h1>
 +
<p>
 +
<h2>Over night cultures</h2>
 +
Start cultures of Gibson Assemblies and J23100<br>
 +
<br>
 +
 +
 +
<h1>15/08/31</h1>
 +
<p>
 +
<h2>Miniprep</h2>
 +
Prep the cultures of pILS7-9, J23100<br>
 +
<br>
 +
 +
<h2>Sequencing</h2>
 +
Send pILS7-9 for sequencing:<br>
 +
Constructs: 2.7.2, 2.8.2, 2.9.3, 3.7.1, 3.7.2, 3.8.1, 3.8.3, 3.9.2, 3.9.3<br>
 +
<br>
 +
 +
 +
<h2>PCR</h2>
 +
Fuse two primers to promoter by PCR<br>
 +
</table>
 +
 +
<style type="text/css">
 +
    /* #header {width:100%;background-color:#f66300;text-align:left;} */
 +
    #layouttable{width:100%; text-align:center;}
 +
    #layouttable td.col{vertical-align:top;text-align:center;}
 +
</style>
 +
</head>
 +
 +
 +
  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
 +
  <tr style="background-color:#f66300;">
 +
        <td></td>
 +
        <td>  Parts </td>
 +
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>5ul</td>
 +
        <td class='col'>iILS30 </td>
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>5ul</td>
 +
        <td class='col'>iILS31 </td>
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>2ul</td>
 +
        <td class='col'>Polymerase</td>
 +
    </tr>
 +
    </tr>
 +
        <td class='col'>4ul</td>
 +
        <td class='col'>dNTPs</td>
 +
    </tr>
 +
    </tr>
 +
        <td class='col'>10ul</td>
 +
        <td class='col'>buffer</td>
 +
    </tr>
 +
    </tr>
 +
</table><br>
 +
<br>
 +
 +
<h2>PCR Purification</h2>
 +
Purify the fragment of the Primer PCR<br>
 +
<br>
 +
 +
 +
<h2>CPEC</h2>
 +
</table>
 +
 +
<style type="text/css">
 +
    /* #header {width:100%;background-color:#f66300;text-align:left;} */
 +
    #layouttable{width:100%; text-align:center;}
 +
    #layouttable td.col{vertical-align:top;text-align:center;}
 +
</style>
 +
</head>
 +
 +
 +
  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
 +
  <tr style="background-color:#f66300;">
 +
        <td></td>
 +
        <td>  10.1 </td>
 +
        <td>10.3</td>
 +
        <td>  11.1 </td>
 +
        <td>11.3</td>
 +
        <td>  12.1 </td>
 +
        <td>  12.3 </td>
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>BB</td>
 +
        <td class='col'>0,7ul </td>
 +
        <td class='col'>0,7ul</td>
 +
        <td class='col'>0,65ul </td>
 +
        <td class='col'>0,65ul</td>
 +
        <td class='col'>1,14ul </td>
 +
        <td class='col'>1,14ul</td>
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>RFP</td>
 +
        <td class='col'>0,32ul </td>
 +
        <td class='col'>0,32ul</td>
 +
        <td class='col'>0,208ul </td>
 +
        <td class='col'>0,208ul</td>
 +
        <td class='col'>0,26ul </td>
 +
        <td class='col'>0,26ul</td>
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>Promoter</td>
 +
        <td class='col'>0,32ul </td>
 +
        <td class='col'>0,46ul</td>
 +
        <td class='col'>0,32ul </td>
 +
        <td class='col'>0,46ul</td>
 +
        <td class='col'>0,32ul </td>
 +
        <td class='col'>0,46ul</td>
 +
    </tr>
 +
    </tr>
 +
        <td class='col'>Terminator</td>
 +
        <td class='col'>3,81ul </td>
 +
        <td class='col'>3,81ul</td>
 +
        <td class='col'>3,81ul </td>
 +
        <td class='col'>3,81ul</td>
 +
        <td class='col'>3,81ul </td>
 +
        <td class='col'>3,81ul</td>
 +
    </tr>
 +
    </tr>
 +
        <td class='col'>H2O</td>
 +
        <td class='col'>28,85ul </td>
 +
        <td class='col'>28,71ul</td>
 +
        <td class='col'>28,94ul </td>
 +
        <td class='col'>28,81ul</td>
 +
        <td class='col'>28,48ul </td>
 +
        <td class='col'>28,34ul</td>
 +
    </tr>
 +
    </tr>
 +
        <td class='col'>dNTPS</td>
 +
        <td class='col'>4ul </td>
 +
        <td class='col'>4ul</td>
 +
        <td class='col'>4ul </td>
 +
        <td class='col'>4ul</td>
 +
        <td class='col'>4ul </td>
 +
        <td class='col'>4ul</td>
 +
    </tr>
 +
    </tr>
 +
        <td class='col'>Buffer</td>
 +
        <td class='col'>10ul </td>
 +
        <td class='col'>10ul</td>
 +
        <td class='col'>10ul </td>
 +
        <td class='col'>10ul</td>
 +
        <td class='col'>10ul </td>
 +
        <td class='col'>10ul</td>
 +
    </tr>
 +
    </tr>
 +
        <td class='col'>Polymerase</td>
 +
        <td class='col'>2ul </td>
 +
        <td class='col'>2ul</td>
 +
        <td class='col'>2ul </td>
 +
        <td class='col'>2ul</td>
 +
        <td class='col'>2ul </td>
 +
        <td class='col'>2ul</td>
 +
    </tr>
 +
    </tr>
 +
</table><br>
 +
<br>
  
 
<h1>TEXT</h1>
 
<h1>TEXT</h1>

Revision as of 17:34, 12 September 2015

15/04/22

Primer Design

Primer Design iILS1-9


15/04/26

Miniprep



15/06/03

PCR

PCR Mix PCR1 PCR2 PCR3 PCR4 PCR5 PCR6
0,5ul BB1 3108 GFP1 3504 BB2 3109 GFP2 3504 BB3 111 GFP3 3504
1ul ILS2 ILS4 ILS2 ILS7 ILS2 ILS9
1ul ILS3 ILS5 ILS6 ILS5 ILS8 ILS5
31,5ul H2O H2O H2O H2O H2O H2O
10ul Buffer Buffer Buffer Buffer Buffer Buffer
4ul dNTPs dNTPs dNTPs dNTPs dNTPs dNTPs
2ul GXL Polymerase GXL Polymerase GXL Polymerase GXL Polymerase GXL Polymerase GXL Polymerase

Cycler Protocol (30x) PCR1,3,5 PCR2,4,6
98°C 10s 10s
55°C 15s 15s
68°C 22s 10s
end cycle
10°C store store


PCR

Repeat PCR of BB1, BB2
Comments: unsatisfied yield for PCR1, PCR3 -> pcr of pcr (3', 4') and repeat PCR with original template (1',2')

Gel extraction

Gel extraction of PCR2,4,6

15/06/04

PCR

PCR of BB111 (BB3)

15/06/08

Gel electrophoresis

PCR1'-4'
ILS
Figure 1: PCR1, 2, 3, 4


PCR

PCR of GFP (PCR2,4,6)

Gel electrophoresis

PCR2,4,6
ILS
Figure 2: PCR1, 2, 3


15/06/09

PCR

Repeating PCR of PCR1, PCR3, PCR4

Gel extraction

Gel of PCR1,3,4
ILS
Figure 3: PCR1, 3, 4
PCR1 correct (longer band is the one)
PCR3??


DpnI digest

Add 2ul of DpnI to the PCR, incubate for 30min @37°C

Gibson Assembly

Gibson1: BB1+GFP1= pILS1
Gibson3: BB3+GFP3= pILS3

Gibson 1 Gibson 2
BB 1,4ul 1,5ul
GFP 3,23ul 2,6ul
H2OC 5,4ul 5,9ul
Gibson MM 10ul 10ul


Transformation

Transform 2ul Gibson mix of Gibson 1 and Gibson3 into NEB electrocompetent cells
Recover 1h, plate 100ul

15/06/11

CPEC1

CPEC 1 CPEC 3
BB 3,34ul 3,78ul
GFP 0,69ul 0,56ul
H2OC 5,97ul 5,65ul


Analytical digest

BB2 4ul
H2O 3ul
XbaI 1ul
SacI 1ul
Cutsmart 1ul

30min @37°C

Gel electrophoresis

Check the digest of BB2
ILS
Figure 4: Problem: Just one band visible; repeat with other enzymes and load undigested BB


15/06/12

Analytical digest of BB2

Digest 1 Digest 2 Digest 3
BB2 4ul BB2 7ul BB2 4ul
H2O 3ul H2O 2ul
PvuII 1ul EcoRI 1ul EcoRI 1ul
PstI 1ul PstI 1ul
Cutsmart 1ul Cutsmart 1ul Cutsmart 1ul

Incubate 30min @37°C

Gel elxtraction

Check the different digests of BB2
ILS
Figure 5: undigested - digest 1 - digest 2 - digest 3
Something is wrong - they don'r show the correct size at all!!! Check registry and wells


15/06/13

Transformation

Transform following plasmids into NEB Turbo (Chemical competent):
K823005 Promoter
K823008 Promoter
K823013 Promoter
Gibson 1
Gibson3
CPEC1
CPEC3

15/06/15

Inoculation

K823005, K823008 & K823013 picked & inoculated into LB-medium
Incubated @ 37 °C, 250 rpm

Miniprep

Prep the Colonies of the ON

From now on:
K823005: BB04
K823008: BB05
K823013

15/06/17

PCR

PCR9 PCR10
0,5ul template BB K8230008 GFP4 (from PCR6)
1ul Primer iILS8 iILS9
1ul Primer iILS2 iILS5
10ul buffer buffer
4ul dNTPs dNTPS
2ul Polymerase Polymerase
31,5ul H2O H2O



Gel electrophoresis

Gel of Digest of the 06/16
Lane1: GFP04, Lane 2: BB08

Gelextraction

Extraction of BB08 and GFP04

15/06/18

CPEC

CPEC2 (pILS5)
CPEC2
2,5ul BB08
4,29ul GFP04
27,21 H2O
10ul buffer
4ul dNTPs
2ul Polymerase


15/06/24

PCR

PCR for pILS4 and pILS6
PCR Mix PCR7 PCR8 PCR11 PCR12
1ul BB05 GFP BB13 GFP
1ul Primer iILS2 Primer iILS5 Primer iILS18 Primer iILS5
1ul Primer iILS16 Primer iILS15 Primer iILS115 Primer iILS17
31ul H2O H2O H2O H2O
10ul buffer buffer buffer buffer
4ul dNTPS dNTPS dNTPS dNTPS
2ul Polymerase Polymerase Polymerase Polymerase


Gel electrophoresis

Load the PCR on a gel to cut out

Gel extraction

Extract all PCRs

15/06/29

CPEC

pILS4 pILS6
BB04 0,2ul BB06 0,8ul
GFP4 1,2ul GFP6 0,9ul
H2O 32,62ul H2O 32,35ul
buffer 10ul buffer 10ul
dNTPs 4ul dNTPs 4ul
Polymerase 4ul Polymerase 4ul

Next day: Transformation - no colonies grew

15/07/01

PCR

PCR for pILS5
PCR Mix PCR9 PCR10
0,5ul BB08 GFP
1ul iILS8 iILS9
1ul iILS2 iILS5
31,5ul H2O H2O
10ul buffer buffer
4ul dNTPs dNTPs
2ul Polymerase Polymerase


PCR

PCR of GFP4, GFP6, BB4, BB6
Gel showes that BB6 was not succesful

Gel extraction

extract BB4, GFP4, GFP6 from gel

PCR

Repeat PCR of BB6

15/07/02

CPEC5, CPEC4

CPEC5 CPEC4
BB4 3,5ul BB04 1,9ul
GFP5 5,1ul GFP4 2,2ul
H2O 25,4ul H2O 29,9ul
buffer 10ul buffer 10ul
dNTPs 4ul dNTPs 4ul
Polymerase 4ul Polymerase 4ul


Transformation

Transform CPECS into NEB (chemically)
no colonies grew

15/07/06

CPEC5, CPEC4

CPEC5 CPEC4
BB4 3,5ul BB04 1,9ul
GFP5 5,1ul GFP4 2,2ul
H2O 25,4ul H2O 29,9ul
buffer 10ul buffer 10ul
dNTPs 4ul dNTPs 4ul
Polymerase 4ul Polymerase 4ul

use different cycler protocol

15/07/07

Transformation

Transform CPEC4, 5 into NEB turbo (chemical transformation)

Glycerol stocks

Prepare glycerol stocks (siGEM08, -siGEM11) of pILS4,5,6 of overnight cultures

Miniprep

Miniprep of Overnight Cultures

PCR

Repeat PCR7-12

15/07/08

PCR

PCR of 7-12
PCR7,8,9,10,12 were successful
Repeat PCR11

Gel extraction

Extract PCRS that worked

CPEC

CPEC4 CPEC6
BB4 0,41ul BB06 0,37ul
GFP4 4,23ul GFP6 9,26ul
H2O 29,35ul H2O 24,37ul
buffer 10ul buffer 10ul
dNTPs 4ul dNTPs 4ul
Polymerase 4ul Polymerase 4ul

Transformation

Transform PEC5 and CPEC6 of the 6th into NEB chem comp. Use 5ul of DNA.
No colonies for CPEC4 the next day
Colonies for CPEC5.

Inoculation

Inoculate 8 colonies of CPEC5 in 5ml LB-CAM and incubate @37°C for 6h.

Minirep

Prep the cultures.

Analytical digest

Digest the 8 plasmids with PvuII

15/07/10

Gel extraction

Extract PCR BB6, BB4, GFP4 and GFP6 out of Gel

CPEC

CPEC4 CPEC6
BB4 1,25ul BB06 8ul
GFP4 0,42ul GFP6 0,42ul
H2O 32,33ul H2O 31,58ul
buffer 10ul buffer 10ul
dNTPs 4ul dNTPs 4ul
Polymerase 4ul Polymerase 4ul



PCR

New PCR of BB6

Gel extraction

Extract BB6 BB6

CPEC

CPEC4 CPEC6
BB4 2,4ul BB06 1,3ul
GFP4 0,7ul GFP6 0,8ul
H2O 30,9ul H2O 31,9ul
buffer 10ul buffer 10ul
dNTPs 4ul dNTPs 4ul
Polymerase 4ul Polymerase 4ul



15/07/11

Transformation

Transform all previous CPECs into NEB turbo, chemical competent cells

15/07/12

Inoculation

Pick 8 colonies of each construct and grow them in LB CAM

15/07/13

Miniprep

Prep all the colonies from construct 4 and 6

Analytical digest

Digest with PvuII
ILS
Figure 6: Lower bands look ok, but somehow there are additional long bands. Chromosomal DNA? Repeat Miniprep


15/07/15

Miniprep

Repeat Miniprep of colony 1-8 of construct 4 and 6

Digest

Repeat digest with PvuII

Overnight Cultures

Start ONC of 6.1 and 6.6

15/07/16

Digest

New digest with new Minipreps with PvuII
ILS
Figure 7: Loaded also undigested plasmid; still weird long bands


ILS
Figure 8: Loaded also undigested plasmid; still weird long bands

ILS
Figure 9: Loaded also undigested plasmid; still weird long bands


Over night cultures

Start ONC of pILS5 for glycerol stock, 4.2, 4.4, 4.7, 6.1, 6.5

15/07/17

Glycerol Stock

Prepare a glycerol stock of pILS5 (sIGEM20)and pILS4 (sIGEM21)

Transformation

Transform pILS4 into Dh5alpha

Miniprep

Prep 6.1 and 6.6

Digest

Analytical digest of 6.1 and 6.6 with PvuII

Sequencing

Send 6.1 and 6.6 for sequencing

15/07/20

Transformation

Transform pILS4,5,6 into wt3110 and MG1655

15/07/21

Over night culture

Pick colonies from pILS4,5,6 in wt3110 and MG1655 and start cultures

15/07/22

Glycerol Stocks

WT3110: pILS4-6 - sIGEM 25-27
MG1655: pILS4-6 - sIGEM 28-31

15/07/27

Restreak all constructs in all strains on plates from glycerol stock

lalala

15/08/11

Transformation

Transformation of E1010 and J23100 into NEB turbo
J23100 wasn't succesful

15/07/12

FACS

Measurement of 3 biological replicats and 3 technical replicats
t= dilution
0h -
1h -
2h 1:10
3h 1:100
4h 1:100
5h 1:100
6h 1:100
6h 1:100


15/07/13

Transformation

Repeat Transformation of J23100 in NEB turbo

15/07/17

Transformation

Repeat Transformation of J23100 in NEB turbo

PCR

PCR of BB7, BB8, BB9, BB10, T10, BB11, T11, BB12, T12

Over night culture

ONC of E1010, pILS4, pILS5, pILS5 in DH5alpha

15/08/18

Miniprep

Prep the cultures of E1010, pILS4, pILS5 and pILS6

PCR

PCR of RFP7, RFP8, RFP9, RFP10, RFP11, RFP12

Gel of PCRS

ILS
Figure 10: Weird bands

DpnI digest

Add 2 ul of DpnI to each PCR tube, incubate for 30min @37°C

PCR Purification

Purify BB7-9 and RFP7-9
Low in concentration afterwards

Gel extraction

Extraction of BB10, RFP10, Term10, BB11, RFP11, Term11, BB12, RFP12, Term12
Low concentrations: repeat PCRs

PCR

Repeat PCRs of BB7-12, RFP7-12, T10-12

Analytical gel of BB7, RFP7, BB8, RFP8, BB9, RFP9

lalala

DpnI digest

Add 2 ul of DpnI to each PCR tube, incubate for 30min @37°C

Transformation

Transform J23100 into electrocompetent cells

15/08/19

PCR Purification

Purify BB7, RFP7, BB8, RFP8, BB9, RFP9

Gibson Assembly

15min @50°C
BB RFP
7 0,58ul 0,91ul
8 0,8ul 0,87ul
9 0,68ul 0,74


Transformation

Transform the Gibson Assemblies into NEB turbo

PCR

PCRs of BB10-12, RFP10-12 and Term 10-12

Gel extraction

Extract BB10, RFP10, Term 10, BB11, RFP11, Term 11, BB12, RFP12, Term 12

Over night culture

Start cultures of pILS4-6 in DH5alpha and MG1655

15/08/20

Platereader experiment

Grow pILS4-6 in MG1655 and DH5alpha in M9 inplater reader
Starting OD 0,01 in 96well plate, measure OD and GFP every 15min

Over night cultures

Start cultures of pILS7.1-7.8, pILS7.1-8.8, pILS7.1-9.8 and J23100

Transformation

Transform pILS9 into NEB turbo

15/09/21

Miniprep

Prep the cultures of pILS7.1-7.8, pILS8.1-8.8 and pILS9.1

PCR

Make the PCRS of Promoter 10-12

Gel

no bands visible at expected length

15/08/22

Digest

Digest pILS7.1-7.8, pILS8.1-8.8 and pILS9.1 with PvuII
ILS
Figure 11: pILS7.1, 8.3 and 9.1 look promising, send for sequencing


15/08/24

Over night cultures

Start cultures of pILS9.2, pILS9.3, pILS9.4 and pILS9.5

Transformation

Transform 0040, I20270 into DH5alpha and pILS9 (Gibson) in NEB turbo

15/08/25

Restreak from glycerol stock

Restreak 0040, I0270, pILS4, pILS5, pILS6 and DH5alpha on plates from glycerol stock - incubate for 16h

Digestion of Promoter/h2> Digest Promoter 10-12 with PvuII and EcoRI<

Gel extraction

extract the band

PCR

make a PCR on gel extracted digested promoter
- no bands visible

15/08/26

Over night cultures

Start cultures (8 biological replica) of 0040, I0270, pILS4, pILS5, pILS6 and DH5alpha

Sequencing

Sequencing of pILS7-9 showed point mutations

15/08/27

Measurement with platereader for ILS submission

Dilution of each culture to OD of 0.5 (platereader) and measurement of fluorescence intensities

15/08/28

Measurement with platereader for ILS submission

Dilution of each culture to OD of 0.5 (cuvette) and measurement of fluorescence intensities

15/08/29

PCR

Amplify parts for pILS7-9

PCR Purification

Purification of the PCRs

Gibson Assembly

Assembly of pILS7-9

Transformation

Transform Gibson Assembly into NEB Turbo

15/08/30

Over night cultures

Start cultures of Gibson Assemblies and J23100

15/08/31

Miniprep

Prep the cultures of pILS7-9, J23100

Sequencing

Send pILS7-9 for sequencing:
Constructs: 2.7.2, 2.8.2, 2.9.3, 3.7.1, 3.7.2, 3.8.1, 3.8.3, 3.9.2, 3.9.3

PCR

Fuse two primers to promoter by PCR
Parts
5ul iILS30
5ul iILS31
2ul Polymerase
4ul dNTPs
10ul buffer


PCR Purification

Purify the fragment of the Primer PCR

CPEC

10.1 10.3 11.1 11.3 12.1 12.3
BB 0,7ul 0,7ul 0,65ul 0,65ul 1,14ul 1,14ul
RFP 0,32ul 0,32ul 0,208ul 0,208ul 0,26ul 0,26ul
Promoter 0,32ul 0,46ul 0,32ul 0,46ul 0,32ul 0,46ul
Terminator 3,81ul 3,81ul 3,81ul 3,81ul 3,81ul 3,81ul
H2O 28,85ul 28,71ul 28,94ul 28,81ul 28,48ul 28,34ul
dNTPS 4ul 4ul 4ul 4ul 4ul 4ul
Buffer 10ul 10ul 10ul 10ul 10ul 10ul
Polymerase 2ul 2ul 2ul 2ul 2ul 2ul


TEXT

Text

lalala

15/09/02

Transformation

Trafo of constr. 10,4+11,4+12,4 in NEB Turbo

PCR

PCR of P30+31

PCR Cleanup

PCR-Clean up of P30+31

CPEC

CPEC of construct 5