Team:Heidelberg/Notebook/Killing I/Notebook/week10

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**pcr was not successful, gel included no pcr product
**pcr was not successful, gel included no pcr product
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+
=== Characterize of oriT===
 +
*Quantitatively test for oriT
 +
Donor: overnight culture Cotransformation Top10 J01103+pUB307(12) OD(600nm): 2.844<br>
 +
Recipient: overnight culture Top10 pBAD 33  OD(600nm): 3.346<br>
 +
**Centrifuge 250ul overnight culture in 1.5ml eppi for 2min at 13000rpm, 10samples donor, 10samples recipient
 +
**Wash the pellet twice with LB medium
 +
**Resolve the pellet in 250ul LB medium
 +
**Centrifuge the washed recipient for 2min at 13000rpm, discard the fluid
 +
**Add the washed donor suspension
 +
**Vortex and resolve the pellet
 +
**Centrifuge the mix for 1min at 13000rpm
 +
**Resolve the pellet in 100ul LB
 +
**Put membrane filter on the LB agar
 +
**Pipett the suspension on membrane filter (10samples)
 +
**Incubate the plates with membrane filter at 37°C
 +
**Put directly one membrane filter into 1ml LB in an 1.5ml eppi
 +
**Vortex the eppi for 30sec, dilute for 10-4 and 10-5, plate them out on LB/Amp+Cm plates (0min)
 +
**After 6, 12, 18, 24, 30, 36, 42, 48, 54min repeat the last two steps.<br>
 +
**Negative control plates:
 +
***LB/Cm+Amp:
 +
100ul donor overnight culture<br>
 +
100ul recipient overnight culture<br>
 +
**Cell number determination
 +
***LB/Cm: 100ul 10-6 recipient overnight culture
 +
***LB/Kan+Amp: 100ul 10-6 donor overnight culture
 +
<br>
 +
*Result:
 +
**Negative control: negative
 +
**Colony on LB/Cm: 324 (Titer of recipient: 3.24e9/ml)
 +
**Colony on LB/Kan+Amp: 373 (Titer of donor: 3.73e9/ml)
 +
**Colony on other LB/Cm+Amp plates:
 +
**10-4 dilute: impossible for counting
 +
**10-5 dilute:<br>
 +
{| class="wikitable"
 +
|- bgcolor=grey
 +
! height=20px. width=200px | Time || width=250px | Colony
 +
|-align="center"
 +
| style="font-weight:bold;" |0 || 150
 +
|-align="center"
 +
| style="font-weight:bold;" |6 || 500
 +
|-align="center"
 +
| style="font-weight:bold;" |12 || 780
 +
|-align="center"
 +
| style="font-weight:bold;" |18 || 1160
 +
|-align="center"
 +
| style="font-weight:bold;" |24 || 1400
 +
|-align="center"
 +
| style="font-weight:bold;" |30 || 3360
 +
|-
 +
|}
 +
***36min to 54 min: impossible for counting
 +
<br>
 +
*Make glycerol stock for Cotransformation Top10 oriT+pUB307
 +
**1ml overnight culture of Top10 oriT+pUB307 (12)+ 150ul 80% glycerol
 +
**Vortex
 +
**1hr RT
 +
**Freeze at -80°C
==Thursday, 10/09/08 ==
==Thursday, 10/09/08 ==

Latest revision as of 14:09, 29 October 2008

<< Week 9 Overview Week 11 >>


Week 10

Contents

Monday, 10/06/08

Cloning of CmR in pBluescript

Proceeding of the overnight ligations

  • 8 transformation of the CmR overnight ligations
  • 3 control transformation of Term1, CmR3 and pBluesript backbone


Proceeding of the minipreps from friday

  • digestions of CmR in pBlue Miniprep to check for sequencing
    • digestion with SacI, KpnI and EcoRI
      • expected bands: 2859, 586, 272
    • digestion with DraI
      • expected bands: 1480, 1187, 692, 339, 19
  • Gel

Hd-phage-08-10-6-digestion-cmr.jpg

    • top:
    • lane1-3: CmR1 (undigested, SacI/KpnI/EcoRI, DraI)
    • lane4-6: CmR2
    • lane7-10: CmR3a
    • lane11-13: CmR3b
    • bottom
    • lane1-3: CmR4 (undigested, SacI/KpnI/EcoRI, DraI)
    • lane4-6: CmR5
    • lane7-10: CmR7
    • lane11-13: CmR8
  • according to the digestions we have the chloramphenicol resistance cassette in pBluescript
  • Retrafo of CmR minipreps because we don't have enough DNA and unfortunately no glycerol stocks
    • 3µl of CmR 1, 2, 3a, 3b

Cloning of CmR in standard plasmid

  • PCR of CmR with CmR_prefix_fw and CmR_suffix_fw 
25µl Phusion Master Mix 2x
1µl CmR_suffix_fw
1µl CmR_prefix_rev
1µl Maxiprep pBlue with insert (stock: ~200ng/µl, dilution: 1:20-->10ng)
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
  • Gel

Hd-phage-08-10-6-cmr-pcr.jpg

    • expected size: 851bp+43bp=894bp
    • lane0:ladder
    • lane1: CmR1
    • lane2: CmR2
    • lane3: CmR3a
    • lane4: CmR3b
    • lane5: CmR4
    • lane6: CmR5
    • lane7: CmR7
    • lane8: CmR8
    • cuted out CmR band and gel extraction kit



Cloning of cI in standard plasmid

  • unwanted EcoRI restriction site in cI from geneart was mutated using standard protocol

Hd-phage-08-10-07 digestion cI EcoRI.jpg

  • lane 1+2: mutated cI digested with EcoRI --> mutagenesis was succesfull
  • maxiprep of this cI as well as J01003 in pSB1A2 were digested with EcoRI/PstI and XbaI/PstI to ligate this mutated cI in a standard plasmid (pSB1A2)

Hd-phage-08-10-07 cutting out cI and pSB1A2 backbone.jpg

  • lane 1-4: cI digested with EcoRI/PstI
  • lane 5-9: cI digested with XbaI/PstI (lane 7 is ladder)
  • lane 10+11: J01003 digested with XbaI/PstI
  • lane 12+13: J01003 digested with EcoRI/PstI

--> digested cI and pSB1A2 backbone was cut out and ligated (15 min, RT) --> transformation in E.coli Top10 --> inoculation of liquid cultures --> miniprep --> send for sequencing and controll digest with EcoRI/PstI

  • seqeuncing was succesfull
  • digestion with EcoRI/PstI

Hd-phage-08-10-20 digestion cI EcoRI-PstI.jpg

  • lane 1-6: mutated cI in pSB1A2 digested with EcoRI/PstI

results: sequencing and digestion pattern is correct


Characterization of oriT

  • Inoculate the cells for conjugation test
    • 5ml LB/chloramphenicol + glycerol stock Top10 pBAD 33
    • 5ml LB/kanamycin/ampicilin + 1 colony Cotransformation Top10 J01103+pUB307(12)
    • 5ml LB/kanamycin/ampicilin + 1 colony Cotransformation Top10 J01103+pUB307(22)
    • 5ml LB/kanamycin/ampicilin + 1 colony Cotransformation Top10 J01103+pUB307(13)
    • 5ml LB/kanamycin/ampicilin + 1 colony Cotransformation Top10 J01103+pUB307(23)

Tuesday, 10/07/08

Proceeding of cloning CmR in pBluescript

  • a lot of single colonies of the transformation of the new ligations
  • a lot of colonies on the retrafo
    • inoculation of liquid culture of the 4 retrafos and the first 4 new ligations
    • agar plates with new ligations are stored at 4 °C

Cloning of CmR in standard plasmid

  • Digestion of CmR pcr product with PstI, XbaI 
5µl NEB3
5µl BSA
10µl PCR product CmR (app. 50ng/µl (from gel) )
1 µl PstI
1,5µl XbaI
32,5µl water
    • 4 samples: 1,2, 3a, 3b
    • 2h at 37°C
    • PCR purification kit
  • ligation of digested CmR pcr product in pSB1A2, 30min at room temperature
  • transformation in TOP10, plated out on CmR plates
  • ligation was done overnight as well


Phage cloning strategy two

  • Digestion of KpnI mutagenesis PCR 
3µl DNA
5µl NEB1
5µl BSA
1µl KpnI
1µl AgeI
36µl water
    • 4 digestions with following mutagenesis pcr samples: 1,3,4,6
  • Gel
    • mutagensis pcr successful: 1690bp, 5050bp
    • -->cut out 5050bp band
    • mutagensis pcr unsuccessful: 1684bp, 1690bp, 3366bp

Hd-phage-08-10-7-pblue-insert.jpg Hd-phage-08-10-7-pblue-insert2.jpg

    • lane0: DNA ladder mix
    • lane1-3: digested pcr sample 1
    • lane4-6: digested pcr sample 3
    • lane7-9: digested pcr sample 4
    • lane10-12: digested pcr sample 6


  • Digestion of CmR out of CmR in pBluescript with KpnI SacI 
5µl NEB1
5µl BSA
3µl CmR miniprep
1,5µl KpnI
1,5µl SacI
34µl water

--> digestion of CmR miniprep 1,2,4,5

  • Gel
    • expected: 858bp, 2859bp
    • -->cut out 858bp band
    • lane0:ladder
    • lane1-3: digested miniprep 1
    • lane4-6: digested miniprep 2
    • lane7-9: digested miniprep 4
    • lane10-12: digested miniprep 5
  • ligation of CmR (KpnI/SacI), GFP (SacI/AgeI), pBluescript (KpnI/AgeI) 30min at room temperature
    • transformation in TOP10
  • ligation was done at 16°C over night as well

Characterization of oriT

  • Qualitatively test for oriT

Donor: overnight culture Cotransformation Top10 oriT+pUB307(12), (22), (13), (23)
Recipient: overnight culture Top10 pBAD 33

    • Centrifuge 500ul overnight culture in 1.5ml eppi for 2min at 13000rpm
    • Wash the pellet twice with LB medium
    • Resolve the pellet in 500ul LB medium
    • Mix 500ul washed recipient cell suspension with 500ul washed donor cell suspension in 2ml eppi
    • Vortex
    • Incubate the mix at 37°C for 1hr
    • Plates:
      • LB/Cm: 100ul overnight culture Top10 pBAD 33
      • LB/Kan+Amp:

100ul overnight culture Top10 oriT+pUB307(12)
100ul overnight culture Top10 oriT+pUB307(22)
100ul overnight culture Top10 oriT+pUB307(13)
100ul overnight culture Top10 oriT+pUB307(23)

      • LB/Amp+Cm:

100ul overnight culture Top10 oriT+pUB307(12)
100ul overnight culture Top10 oriT+pUB307(22)
100ul overnight culture Top10 oriT+pUB307(13)
100ul overnight culture Top10 oriT+pUB307(23)
100ul overnight culture Top10 pBAD 33
100ul conjugation mix (12)
100ul conjugation mix (13)
100ul conjugation mix (22)
100ul conjugation mix (23)

  • Result:

All LB/Cm positive; all LB/Kan+Amp positive; LB/Amp+Cm with donor or recipient negative; LB/Amp+Cm with conjugation mix positive -> like expectation

  • Inoculate cells for conjugation test
    • 5ml LB/chloramphenicol + 10ul overnight culture Top10 pBAD 33
    • 5ml LB/kanamycin/ampicilin + 10ul overnight culture Cotransformation Top10 oriT+pUB307(12)
    • 5ml LB/kanamycin/ampicilin + 10ul overnight culture Cotransformation Top10 oriT+pUB307(22)
    • 5ml LB/kanamycin/ampicilin + 10ul overnight culture Cotransformation Top10 oriT+pUB307(13)
    • 5ml LB/kanamycin/ampicilin + 10ul overnight culture Cotransformation Top10 oriT+pUB307(23)

Wednesday, 10/08/08

Phage cloning strategy two

  • transformation of overnight ligations in TOP10
  • screening pcr to check if ligation was successful using GFP_new_fw and GFP_new_rv
    • pcr was not successful, gel included no pcr product

Characterize of oriT

  • Quantitatively test for oriT

Donor: overnight culture Cotransformation Top10 J01103+pUB307(12) OD(600nm): 2.844
Recipient: overnight culture Top10 pBAD 33 OD(600nm): 3.346

    • Centrifuge 250ul overnight culture in 1.5ml eppi for 2min at 13000rpm, 10samples donor, 10samples recipient
    • Wash the pellet twice with LB medium
    • Resolve the pellet in 250ul LB medium
    • Centrifuge the washed recipient for 2min at 13000rpm, discard the fluid
    • Add the washed donor suspension
    • Vortex and resolve the pellet
    • Centrifuge the mix for 1min at 13000rpm
    • Resolve the pellet in 100ul LB
    • Put membrane filter on the LB agar
    • Pipett the suspension on membrane filter (10samples)
    • Incubate the plates with membrane filter at 37°C
    • Put directly one membrane filter into 1ml LB in an 1.5ml eppi
    • Vortex the eppi for 30sec, dilute for 10-4 and 10-5, plate them out on LB/Amp+Cm plates (0min)
    • After 6, 12, 18, 24, 30, 36, 42, 48, 54min repeat the last two steps.
    • Negative control plates:
      • LB/Cm+Amp:

100ul donor overnight culture
100ul recipient overnight culture

    • Cell number determination
      • LB/Cm: 100ul 10-6 recipient overnight culture
      • LB/Kan+Amp: 100ul 10-6 donor overnight culture


  • Result:
    • Negative control: negative
    • Colony on LB/Cm: 324 (Titer of recipient: 3.24e9/ml)
    • Colony on LB/Kan+Amp: 373 (Titer of donor: 3.73e9/ml)
    • Colony on other LB/Cm+Amp plates:
    • 10-4 dilute: impossible for counting
    • 10-5 dilute:
Time Colony
0 150
6 500
12 780
18 1160
24 1400
30 3360
      • 36min to 54 min: impossible for counting


  • Make glycerol stock for Cotransformation Top10 oriT+pUB307
    • 1ml overnight culture of Top10 oriT+pUB307 (12)+ 150ul 80% glycerol
    • Vortex
    • 1hr RT
    • Freeze at -80°C

Thursday, 10/09/08

Cloning of CmR in standard plasmid

  • miniprep of 12 from the 20 overnight cultures (CmR in pSB1A2)
    • sample nr. 1.1-1.3, 2.1-2.3, 3.1-3.3, 4.1-4.3
  • digestion of the 12 minipreps with EcoRI and PstI 
4µl EcoRI buffer
4µl BSA 
1µl EcoRI
1µl PstI
3µl Miniprep DNA
27µl water
  • Gel

Hd-phage-08-10-9-digestionCmRpsB1A2.jpg

  • expected fragments: 292,601,2038bp
    • lane0: DNA ladder mix
    • lane1: 1.1
    • lane2: 1.2
    • lane3: 1.3
    • lane4: 2.1
    • lane5: 2.2
    • lane6: 2.3
    • lane7: 3.1
    • lane8: 3.2
    • lane9: 3.3
    • lane10: 4.1
    • lane11: 4.2
    • lane12: 4.3
  • mutagenesis pcr with 2.2 and 2.3 overnight to remove EcoRI restriction site 
5µl Pfu buffer
2µl CmR_EcoRI_mut_fw
2µl CmR_EcoRI_mut_rv
1,5µl dNTPs
1µl Miniprep, diluted 1:10
1µl Pfu (not turbo!)
37.5µl water

PCR protocol
95°C 30s
95°C 30s    |
55°C 45s    | 16x
68°C 6.5min |
4°C for ever


Phage cloning strategy two

  • screening pcr with CmR_prefix_fw and CmR_suffix_rv primer (using Taq)
  • Gel

Hd-phage-08-10-9-pBlueCmRGFP-screening.jpg

    • lane0: dna ladder
    • lane1: colony 1+2
    • lane2: colony 3+4
    • lane3: colony 5+6
    • lane4: colony 7+8
    • lane5: colony 9+10
    • lane6: colony 11+12
    • lane7: colony 13+14
    • expected sizes:
      • pBlue with insert: 1668bp
      • CmR cassette only: 852bp
      • GFP cassette only: 919bp
  • colonys 1,2,3,4,9,10 look good



Friday, 10/10/08

Cloning of CmR in standard plasmid

  • proceeding of EcoRI mutagenesis pcr
    • 3h DpnI digestion at 37°C
    • transformation in TOP10


  • digestion of the 12 minipreps (CmR in pSB1A2) with PstI/XbaI
    • expected fragments: 2053bp, 878bp
  • Gel

Hd-phage-08-10-10-digestionCmR-pSB1A2.jpg

    • lane0: ladder
    • lane1-12: miniprep 1.1 - 4.3

--> miniprep 1.1, 2.1, 2.2 look good


Phage cloning strategy two

  • Screening PCR wirh oriT_fw and CmR_suffix_rv (using Taq)
    • expected sized:
      • pBlue/insert: 2150bp
      • pBlue/GFP/CmR: ca. 1.3kb

Hd-phage-08-10-10-screening pcr-pBlue.jpg

  • Gel
  • lane0: DNA ladder mix
  • lane1-12: sample 1-12
    • lane 7 looks good ~1.3kb


Saturday, 10/11/08

Cloning of oriT in standard plasmid

  • Miniprep of oriT (from RP4)
  • PCR with oriT prefix/suffix primer using Phusion
    • expected size: ~500bp
    • 4 pcr samples

Hd-phage-08-10-11-oriT-pcr-product.jpg

    • lane0: dna ladder mix
    • lane1-4: oriT pcr probe 1-4
  • PCR purification kit
  • Digestion with PstI/EcoRI
  • PCR purification kit
  • Ligation in pSB1A2 20min at RT (after 40min heat inactivation at 65°C)
  • Transformation
    • transformation was done on Cm plates although there is no Cm resistance (failure!)
    • -->make transformation again on sunday and plate out on Amp plates


Proceeding of cloning CmR in standard plasmid

  • inoculation of Mutagensis PCR 2.2 (Cmr Std) (only one colony was grown)
  • Mutagenesis PCR of 1.1, 1.2 and 2.2 (CmR Std) using Turbo Pfu 
5µl Pfu buffer
2µl CmR_EcoRI_mut_fw
2µl CmR_EcoRI_mut_rv
1,5µl dNTPs
1µl Miniprep, diluted 1:10
1µl turbo Pfu
37.5µl water

PCR protocol
95°C 30s
95°C 30s    |
55°C 45s    | 16x
68°C 7min |
4°C for ever



Phage cloning strategy two

  • inoculation of pBluescript+GFP+CmR 1-6,9,10 (nach fraktioniertem Ausstrich)
  • redo Screening PCR wirh oriT_fw and CmR_suffix_rv (using Taq)
    • no bands visible! (only primers)




Sunday, 10/12/08

Proceeding of cloning CmR in standard plasmid

  • Miniprep of Mutagenesis PCR 2.2
  • Digestion of Mutagenesis PCR Miniprep 2.2 with PstI, EcoRI
    • -->digestion showed that the mutagenesis PCR did not work


  • DpnI digestion of mutagenesesis PCR (2h at 37°C)
  • transformation of mutagenesis PCR in TOP10


Proceeding of cloning oriT in standard plasmid

  • redo transformation and plate out on Amp plates


Phage cloning strategy two

  • Miniprep of pBlue+GFP+CmR 1-6,9,10


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