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Revision as of 12:39, 29 October 2008 (view source) Yincai (Talk | contribs) (→Characterize of oriT) ← Older edit		Latest revision as of 14:09, 29 October 2008 (view source) Yincai (Talk | contribs) (→Characterize of oriT)
Line 740:		Line 740:
	**10-4 dilute: impossible for counting		**10-4 dilute: impossible for counting
	**10-5 dilute:<br>		**10-5 dilute:<br>
-	Time Colony<br>	+	{| class="wikitable"
-	0 150<br>	+	|- bgcolor=grey
-	6 500<br>	+	! height=20px. width=200px | Time || width=250px | Colony
-	12 780<br>	+	|-align="center"
-	18 1160<br>	+	| style="font-weight:bold;" |0 || 150
-	24 1400<br>	+	|-align="center"
-	30 3360<br>	+	| 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		***36min to 54 min: impossible for counting
	<br>		<br>

---

Latest revision as of 14:09, 29 October 2008

<< Week 9	Overview	Week 11 >>

Week 10

Contents 1 Monday, 10/06/08 1.1 Cloning of CmR in pBluescript 1.1.1 Proceeding of the overnight ligations 1.1.2 Proceeding of the minipreps from friday 1.2 Cloning of CmR in standard plasmid 1.3 Cloning of cI in standard plasmid 1.4 Characterization of oriT 2 Tuesday, 10/07/08 2.1 Proceeding of cloning CmR in pBluescript 2.2 Cloning of CmR in standard plasmid 2.3 Phage cloning strategy two 2.4 Characterization of oriT 3 Wednesday, 10/08/08 3.1 Phage cloning strategy two 3.2 Characterize of oriT 4 Thursday, 10/09/08 4.1 Cloning of CmR in standard plasmid 4.2 Phage cloning strategy two 5 Friday, 10/10/08 5.1 Cloning of CmR in standard plasmid 5.2 Phage cloning strategy two 6 Saturday, 10/11/08 6.1 Cloning of oriT in standard plasmid 6.2 Proceeding of cloning CmR in standard plasmid 6.3 Phage cloning strategy two 7 Sunday, 10/12/08 7.1 Proceeding of cloning CmR in standard plasmid 7.2 Proceeding of cloning oriT in standard plasmid 7.3 Phage cloning strategy two

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

• • 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

• • 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

• 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)

• 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

• 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

• • 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

• 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

• • 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

• • 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

• 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

• • 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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