Team:Heidelberg/Notebook/Killing I/Notebook/week9

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Revision as of 10:49, 29 October 2008

Week 9

Back to the overview

1 Monday, 09/29/08
- 1.1 new phage cloning strategy
  - 1.1.1 PCR of GFP (I20260) and terminator (B0015)
  - 1.1.2 Processing of the KpnI Mutagenesis PCR from Friday
- 1.2 Cloning of oriT in standard plasmid
2 Tuesday, 09/30/08
3 Wednesday, 10/01/08
4 Thursday, 10/02/08
5 Friday, 10/03/08
6 Sunday, 10/05/08
- 6.1 Cloning of CmR in pBluescript

Monday, 09/29/08

new phage cloning strategy

PCR of GFP (I20260) and terminator (B0015)

25µl Phusion Master Mix 2x
1µl GFP_new_fw
1µl GFP_new_rev
1µl Maxiprep I20260 (stock: 200ng/µl, dilution: 1:10)
22µl water
-----
50µl

25µl Phusion Master Mix 2x
1µl Term_new_fw
1µl Term_new_rev
1,5µl Miniprep B0015 (Andi, stock: 80ng/µl, dilution: 1:10)
21,5µl water
-----
50µl

PCR protocol
95°C 1min
95°C 30s  |
55°C 30s  | 26x
72°C 1min |
72°C 10min
4°C for ever

each two pcr samples (GFP new 1, GFP new 2, Term new 1, Term new 2)
Gel (1µl DNA)
expected sizes: GFP: 919bp + 28bp = 947bp, Terminator: 129bp + 22bp = 151bp

lane 0: DNA ladder mix
lane 1: Term new 1 (151bp)
lane 2: Term new 2 (151bp)

lane 0: DNA ladder mix
lane 3: GFP new 1 (947bp)
lane 4: GFP new 2 (947bp)

-->PCR seems to be good

Processing of the KpnI Mutagenesis PCR from Friday

DpnI digestion (1µl DpnI to 50µl PCR sample, 2h 37°C)
PCR purification kit (eluted in 30µl water)
Digestion with KpnI (2h 37°C)

2µl NEB1
2µl BSA
1µl DNA
2µl KpnI
13µl water
-----
20µl

nothing can be seen on the gel --> do the digestion again with more template DNA
Digestion with KpnI (1h 37°C)

5µl NEB1
5µl BSA
10µl DNA
3µl KpnI
27µl water
-----
50µl

retrafo with 2µl and 6µl DNA on Amp/Cm plates
no colonies could be seen on the next day

Cloning of oriT in standard plasmid

inoculated colonies from transformation minipreped and digested with sfcI, one sample with NotI
gel showed that we did not have the expected insert
control PCR to verify this with ORiT primer and primer for standard plasmid -> no insert, too
conclusion: start OriT cloning new

OriT PCR with OriT primer with prefix and suffix (oriT_pre, oriT_suf) from pBluescript with insert using Phusion
maxiprep of pSB1A3 with insert (T9002) inoculated
Tomorrow: prep pSB1A3, digest with XbaI, PstI, cut backbone out of the gel, purificate, digest OriT with XbaI, PstI, ligate both and transform
J01003 (oriT from registry) inoculated from glycerol stock to prep tomorrow and cotransform with pub307

Tuesday, 09/30/08

new cloning strategy

Mutagenesis PCR with mut_kpn1_pBlue

5 µl Pfu Buffer 5x
1 µl mut_kpn1_pBlue_fw
1 µl mut_kpn1_pBlue_rev
1,5 µl dNTPs
1 µl template DNA (pBlue with insert dilution: 1:200)
1 µl Pfu turbo
39,5 µl water
------
50 µl

PCR protocol
95°C 30s
95°C 30s   |
55°C 45s   | 16x
68°C 14min |
4°C for ever

4 pcr samples
1µl DpnI to each sample--> 1h at 37°C
PCR purification kit
concentration measurement
transformation in Top10 cells --> plated out on Cm plates

PCR of CmR

25µl Phusion Master Mix 2x
1µl CmR_new_fw
1µl CmR_new_rev
1µl Maxiprep pBlue with insert (stock: ~2µg/µl, dilution: 1:200)
22µl water
-----
50µl

PCR protocol
98°C 1min
98°C 10s  |
61°C 10s  | 26x
72°C 45s  |
72°C 5min
4°C for ever

4 PCR samples
PCR purification kit
concentration measurement
Gel

lane0: DNA ladder mix
lane1-4: 1µl of pcr product (expected size: 738)

Cloning of CmR in pBluescript

digestion of CmR pcr product (80ng/µl) with KpnI and HindIII

5µl NEB2
5µl BSA
10µl CmR
3µl KpnI
1,5µl HindIII
25,5µl water 
-----
50µl

- digestion of the four pcr samples
- PCR purification kit

digestion of Terminator pcr product (30ng/µl) with HindIII and SacI

5µl NEB2
5µl BSA
25µl Terminator
3µl SacI
1,5µl HindIII
10,5µl water 
-----
50µl

- digestion of the two pcr samples
- PCR purification kit

digestion of pBluescript (stock: maxiprep, 280ng/µl) with KpnI and SacI

5µl NEB1
5µl BSA
3µl pBluescript
2µl KpnI
2µl SacI
33µl water
-----
50µl

- digestion 4x
Gel
- two bands: 2,9kb and 100bp
- cutted out 2,9kb band
- gel purification kit (2 samples: pBlue1 (106ng/µl) and pBlue2 (160ng/µl))

Ligation (vector:insert = 1:3)
- each ligation one time with CmR1/Term1 and CmR2/Term2
  - -->8 ligations
- ligations 30min at room temperature

- Ligation (300ng, pBlue1):

pBlue: 1,4 µl
CmR: 1,9 µl
Term: 1 µl
T4 Ligase: 1µl
Buffer: 2µl
water: 12,7µl

- Ligation (200ng, pBlue1):

pBlue: 1 µl
CmR: 1,25 µl
Term: 0,67 µl
T4 Ligase: 1µl
Buffer: 2µl
water: 14,1µl

- Ligation (300ng, pBlue2):

pBlue: 1 µl
CmR: 1,9 µl
Term: 1 µl
T4 Ligase: 1µl
Buffer: 2µl
water: 13,1µl

- Ligation (200ng, pBlue2):

pBlue: 0,6 µl
CmR: 1,25 µl
Term: 0,67 µl
T4 Ligase: 1µl
Buffer: 2µl
water: 14,5µl

transformation of the 8 ligations in TOP10, plated out on Cm plates

Cloning of oriT in standard plasmid

Producing the backbone

maxiprep of T9002 in pSB1A3 (or pSB1A2 - will be sequenced by colicin group)
Digestion with PstI, XbaI, NcoI for 1h (2 samples)

4µl NEB3
4µl BSA
2µl DNA
1µl PstI
1,5µl XbaI
1,5µl NcoI
26µl water
 -----
40µl

run gel and cut out 2000bp fragment, gel purification kit

PCR of oriT

first round of PCR amplification of OriT from pBluescript with insert went rong - no DNA in tubes
second round worked, 3 samples made

25µl Phusion Master Mix 2x
2µl OriT_pref
2µl OriT_suf
1µl Maxiprep pBlue with insert (stock: ~2µg/µl, dilution: 1:200)
20µl water
-----
50µl

PCR protocol
98°C 1min
98°C 30s  |
60°C 30s  | 30x
72°C 45s  |
72°C 5min
4°C for ever

Digestion and ligation

digestion of oriT pcr product with PstI, XbaI

4µl NEB1
4µl BSA
28µl OriT
1,5µl PstI
1,5µl XbaI
1µl water
-----
40µl

check new oriT samples

- lane0: DNA ladder mix
- lane4-6: oriT pcr samples 1-3 (expected size: ~500bp)

ligation: backbone to insert ratio 1:3, ligate for 40 min. at room temperature

4µl vector (backbone of T9002)
2µl insert (oriT PCR product)
1µl T4 Ligase
2µl Ligase Puffer
11µl water
-----
40µl

transformation in TOP10

Conjugation Tests

Conjugation of pUB307 together with

J01003 (2 samples)
I714031 (2 samples)
mix and let conjugate for 1h, plated in Kan/Amp plates

Test of J01003

digestion with NcoI (to cut out OriT-insert) - worked

Wednesday, 10/01/08

Cloning of oriT in standard plasmid

transformation of oriT worked - colonies on all plates
ligation tested with colony PCR - 2 colonies per plate

25µl PCR Master Mix 2x (Taq)
2µl VF2
2µl VR
21µl water

PCR protocol
95°C   5min
95°C   60s   |
50.5°C 60s   | 25x
72°C   60s   |
4°C for ever

gel results: all tested colonies had right insert (~600bp)

top
- lane0: DNA ladder mix
- lane1-7: sample 1-7
bottom
- lane0: DNA ladder mix
- lane1-6: sample 8-14
sample 7 and 12 inoculated for maxiprep

oriT conjugation test

samples 1, 7, 10, 12 conjugated with pUB307: take 2 ml of liquid culure, spin down and discard 1 ml supernatant, resuspend, mix cultures and plate on Kan/Amp

conjugation of pUB307 in J01003 and I714031 worked
liquid cultures for conjugation tests inoculated - did not grow

Lambda phage

colony PCR of two samples (a and b) of last lambda ligation that gave colonies (and have already been minipreped:
two PCR samples from each liquid culure
gel result: expected fragment length: 4kb - all samples were positive!

cI

cI received from geneart today
digestion of cI (geneart) with EcoRI

--> right fragment size

Proceeding of Mutagenesis PCR with mut_kpn1_pBlue

inoculation of 12 overnight cultures of KpnI_mut_pBlue mutagenesis pcr colonies

Thursday, 10/02/08

Cloning of CmR in pBluescript

on all 8 Cm plates of the pBlue+CmR+Terminator ligation colonies could be seen
inoculation of 8 overnight cultures

Proceeding of Mutagenesis PCR with mut_kpn1_pBlue

12 minipreps of KpnI mut1 pcr samples

Digestion with KpnI and AgeI to get the backbone

10µl DNA (~1µg)
2µl NEB1
2µl BSA
1µl KpnI
1µl AgeI
4µl water

12 samples
Gel
expected bands: 1.7kb and 5kb
insert band with about 1.7kb on every sample
5kb and tight 6kb band
cutted out both bands

Digestion of J01003 (oriT) to get the backbone pSB1A2

1µl DNA
5µl NEB3
5µl BSA
1,5µl PstI
2µl XbaI
35,5µl water

4 samples
Gel
2kb and 1kb band
-->cutted out 2kb band and purified

Gel

pBlue Maxi 5.9 (doesn't make sense on gel) pBlue/insert 6kb pBlue/insert 6kb pBlue/insert 5kb pBlue/insert 5kb pSB1A2 2kb pSB1A2 2kb pcr product of insert from J01003 ladder

Friday, 10/03/08

Cloning of CmR in pBluescript

miniprep of the 8 pBlue+CmR+Terminator overnight cultures
concentrations [ng/µl]
- 189
- 216
- 261
- 273
- 264
- 192
- 270
- 239

digestion of the Minipreps with KpnI and SacI

10µl Miniprep DNA
2µl NEB1
2µl BSA
1µl KpnI
1µl SacI
4µl water

Gel

- lane0: DNA ladder mix
- lane1: 1
- lane2: 2
- lane3: 3a
- lane4: 3b
- lane5: 4
- lane6: 5
- lane7: 7
- lane8: 8

- gel overloaded
- one band at about 600bp, should be around 850

- proceeding: PCR with CmR primers, but they can't be found
- proceeding: sequencing of CmR in pBlue
- proceeding: digest again with a smaller amount of Miniprep DNA
- proceeding: digest with other enzymes to check if it's pBlue

Checking the insert of J01003

25µl Phusion Master Mix
1µl StandardFW
1µl StandardRV
1µl Maxiprep DNA (1:100)
22µl water

PCR protocol
95°C 30s
95°C 30s   |
55°C 45s   | 16x
68°C 14min |
4°C for ever

insert has a length about 1,2kb

--> should be about 600bp

maxiprep of two oriTs in pSB1A2 --> both were sequenced and digested with EcoRI/PstI
result: oriT12 is T9002 (from sequencing) but oriT7 fits (digestion and sequencing) even though the sequencing with the forward primer was not succesful.
our J01003 which we got from the MIT seems not to be correct!! After digestion with EcoRI/PstI there should appear a band at ~400bp but it runs at ~1200bp. This result was reproduced twice. Nevertheless it seems to be an oriT since it works succesfull in conjugation tests. We will prep it again next week from the glycerolstock and reepeat the digestion to make sure we did not contaminate it.

new phage cloning strategy

digestion of GFP PCR product (70 ng/µl) with SacI and AgeI

10µl DNA
5µl NEB1
5µl BSA
2µl AgeI
2µl SacI
26µl water

- two digestion
- pcr purification kit

Sunday, 10/05/08

Cloning of CmR in pBluescript

the cloning was done for a second time due to the bad digestion results on friday
digestion of pBluescript with KpnI and SacI

5µl NEB1
5µl BSA
3µl pBluescript
1,5µl KpnI
1,5µl SacI
34µl water
-----
50µl

- two digestions
Gel
Gel extraction kit
8 overnight ligations of pBlue backbone, CmR3/CmR4 and Term1/2
- 16°C, 14h

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+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4,
+div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4 li a {
+  width: 11em; /* Drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5,
+div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5 li a {
+  width: 12em; /* Drop-down menu width. */
+}
+/*...............................................................................................*/
+/*
+Menu Bar 1
+Drop-Down Menu #2
+Side Menu #1
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 {
+  left: 15.5em !important; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+/*
+Menu Bar 1
+Drop-Down Menu #2
+Side Menu #2
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 {
+  left: 15.5em  !important; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+/*
+Menu Bar 1
+Drop-Down Menu #2
+Side Menu #3
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 {
+  left: 15.5em  !important; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+</style>
 <body>
@@ Line 52: / Line 393: @@
 		</li>
 		<li>
-			<a href="https://2008.igem.org/Team:Heidelberg/Chemotaxis" style="color: white">Modeling<!--[if gt IE 6]><!--></a><!--<![endif]-->
+			<a href="https://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling<!--[if gt IE 6]><!--></a><!--<![endif]-->
 			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
 				<ul class="DropDownMenu" id="MB1-DDM3">
@@ Line 62: / Line 403: @@
 		</li>
 		<li>
-			<a href="https://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
+			<a href="https://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
 			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
 				<ul class="DropDownMenu" id="MB1-DDM5">
@@ Line 98: / Line 439: @@
 		</li>
 		<li style="width: 160px">
-			<a href="https://2008.igem.org/Team:Heidelberg/Project/General_information" style="color: white">Human Practice<!--[if gt IE 6]><!--></a><!--<![endif]-->
+			<a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice<!--[if gt IE 6]><!--></a><!--<![endif]-->
 			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
 				<ul class="DropDownMenu" id="MB1-DDM4">
@@ Line 106: / Line 447: @@
                                 <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"><span><span>Surveys</span></span></a></li>
                                 <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"><span><span>Open Day</span></span></a></li>
                                 <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"><span><span>Nobel Prize</span></span></a></li>
 				</ul>
 				<!--[if lte IE 6]></td></tr></table></a><![endif]-->
@@ Line 120: / Line 462: @@
 </body>
 </html>

Team:Heidelberg/Notebook/Killing I/Notebook/week9

From 2008.igem.org

Revision as of 10:49, 29 October 2008

Contents

Monday, 09/29/08

new phage cloning strategy

PCR of GFP (I20260) and terminator (B0015)

Processing of the KpnI Mutagenesis PCR from Friday

Cloning of oriT in standard plasmid

Tuesday, 09/30/08

new cloning strategy

Mutagenesis PCR with mut_kpn1_pBlue

PCR of CmR

Cloning of CmR in pBluescript

Cloning of oriT in standard plasmid

Producing the backbone

PCR of oriT

Digestion and ligation

Conjugation Tests

Wednesday, 10/01/08

Cloning of oriT in standard plasmid

oriT conjugation test

Lambda phage

cI

Proceeding of Mutagenesis PCR with mut_kpn1_pBlue

Thursday, 10/02/08

Cloning of CmR in pBluescript

Proceeding of Mutagenesis PCR with mut_kpn1_pBlue

Digestion with KpnI and AgeI to get the backbone

Digestion of J01003 (oriT) to get the backbone pSB1A2

Friday, 10/03/08

Cloning of CmR in pBluescript

Checking the insert of J01003

new phage cloning strategy

Sunday, 10/05/08

Cloning of CmR in pBluescript