Team:Heidelberg/Templates/DelH week26

From 2013.igem.org

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(→Screening ccdB-construct)
 
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=21-10-2013=
=21-10-2013=
 +
====Amplification of pFS_02 Backbone (HM_17 to FS_78; 4.1 kbp)====
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! Reagent !! Amount [µl] 
 +
|-
 +
| pSB6A1 || 1
 +
|-
 +
| HM_17:  (1/10) || 1
 +
|-
 +
| FS_78:  (1/10) || 1
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| DMSO || 1
 +
|-
 +
| dd H<sub>2</sub>O || 4
 +
|}
 +
 +
:'''Conditions '''
 +
 +
{| class="wikitable"
 +
|-
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! colspan="3" | Biorad T100
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="3"| 35 || 98 || 1
 +
|-
 +
| 58  || 5
 +
|-
 +
| 72  || 1:25
 +
|-
 +
| 1 || 72 || 10min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
 +
====Amplification of Backbone fragment for pFS_02 (HM_17 to FS_87; 4.2 kbp)====
 +
4 reactions with 20µl each, followed by DpnI digest
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! Reagent !! Amount [µl] 
 +
|-
 +
| pSB6A1 || 0.2
 +
|-
 +
| HM_17:  (1/10) || 1
 +
|-
 +
| FS_87:  (1/10) || 1
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| DMSO || 1
 +
|-
 +
| dd H<sub>2</sub>O || 4.8
 +
|}
 +
 +
:'''Conditions'''
 +
 +
{| class="wikitable"
 +
|-
 +
! colspan="3" | Biorad T100
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="3"| 12 || 98 || 1
 +
|-
 +
| 68 ↓ 0.5  || 5
 +
|-
 +
| 72  || 1:25
 +
|-
 +
| rowspan="3"| 18 || 98 || 1
 +
|-
 +
| 66  || 5
 +
|-
 +
| 72  || 1:25
 +
|-
 +
| 1 || 72 || 10min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
 +
 +
==Gibson assembly of pFS_02==
 +
{|class="wikitable"
 +
|-
 +
! Reagent !! Volume added (µl)
 +
|-
 +
| BB (HM_17-FS_78) || 0.24
 +
|-
 +
| DN11-FS66 || 2.13
 +
|-
 +
| FS_67-FS_68 || 2.89
 +
|-
 +
| FS_69-FS_70 || 2.38
 +
|-
 +
| FS_71-HM08 || 2.36
 +
|-
 +
| Gibson Master Mix 2x || 10
 +
|}
 +
 +
====Colony-PCR, screening for pFS_02====
 +
{| class="wikitable" style="float:left; margin-right:1em"
 +
|-
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| Template || 96x 1 picked colony
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|-
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| Expected length [bp] ||  521
 +
|-
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| Named || 1A - 12H, DelH_pSB6A1_weak_promotor
 +
|-
 +
| Primer fw 10 µM ||96x 1 µl FS_73 
 +
|-
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| Primer rev 10 µM ||96x 1 µl FS_95
 +
|-
 +
| One-Taq Polymerase (2x) ||96x 5 µl
 +
|-
 +
| ddH<sub>2</sub>O || 96x 4 µl 
 +
|}
 +
{| class="wikitable" style="float:left; margin-right:1em"
 +
|-
 +
! Cycles !! Temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 95 || 120
 +
|-
 +
| rowspan="3" | 12 || 95 || 30
 +
|-
 +
| 68 (touchdown -0.5°C) || 30
 +
|-
 +
| 72 || 45 s
 +
|-
 +
| rowspan="3" | 18 ||  95 || 30
 +
|-
 +
| 65  || 30
 +
|-
 +
| 72 || 45
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
<div style="clear:both"></div>
 +
 +
10 colonies positive for screening
 +
none of them positive for restriction digest.
 +
==Preparation of large plasmids==
==Preparation of large plasmids==
6 ml of clones DelH+pSB4K5 A1, A4, A7, B4
6 ml of clones DelH+pSB4K5 A1, A4, A7, B4

Latest revision as of 11:08, 26 October 2013

Contents

21-10-2013

Amplification of pFS_02 Backbone (HM_17 to FS_78; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB6A1 1
HM_17: (1/10) 1
FS_78: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
58 5
72 1:25
1 72 10min
1 12 inf

Amplification of Backbone fragment for pFS_02 (HM_17 to FS_87; 4.2 kbp)

4 reactions with 20µl each, followed by DpnI digest

Reaction
Reagent Amount [µl]
pSB6A1 0.2
HM_17: (1/10) 1
FS_87: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4.8
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 1:25
18 98 1
66 5
72 1:25
1 72 10min
1 12 inf


Gibson assembly of pFS_02

Reagent Volume added (µl)
BB (HM_17-FS_78) 0.24
DN11-FS66 2.13
FS_67-FS_68 2.89
FS_69-FS_70 2.38
FS_71-HM08 2.36
Gibson Master Mix 2x 10

Colony-PCR, screening for pFS_02

Template 96x 1 picked colony
Expected length [bp] 521
Named 1A - 12H, DelH_pSB6A1_weak_promotor
Primer fw 10 µM 96x 1 µl FS_73
Primer rev 10 µM 96x 1 µl FS_95
One-Taq Polymerase (2x) 96x 5 µl
ddH2O 96x 4 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45 s
18 95 30
65 30
72 45
1 12 inf

10 colonies positive for screening none of them positive for restriction digest.

Preparation of large plasmids

6 ml of clones DelH+pSB4K5 A1, A4, A7, B4

Restriction Digest of Preps with PvuI

0.5 µl PvuI-HF, 2 µl CutSmart, 1 µl 1:10 Prep (1-2 µg)
2 hours of incubation
expected bands: 11.6 kbp, 5.8 kbp, 4.6 kbp

Results:
--> only clone A1 was positive
--> A1 was send to sequencing with primer DN07

Screening colonies of pSB4K5+DelH (without promotor)

46 colonies
0.5 Primer (FS73 and FS95)
5 µl Dream-Taq
4 µl ddH2O
Results:

  • Colonies B4, B9, B10 and C4 were positive
  • Those colonies were inoculated in 6 ml LB Kan

Test if electrocompetent BL21-DE3 with DelRest and pIK8.6 contain DelRest

Screening of plasmid pFSN-D8w (positive control), Triple clone and electrocompetent BL21-DE3 with DelRest and pIK8.6
0.5 µl FS_14
0.5 µl VR
4 µl ddH2O
5 µl Dream Taq
Results:

  • All three screenings were positive. Consequently our electrocompetent BL21-DE3 with DelRest and pIK8.6 really contain DelRest.

Screening ccdB-construct

14 colonies were picked, we had a negative control was used

Reagent Volume
KH10 0.5 µl
VFII 0.5 µ
ddH2O 4 µl
Dream Taq 5 µl

Annealing temp.: 56°C, 35 cycles, elongation time= 50 s

Results:

  • All screened colonies were positive.
  • Colonies 3, 7, 11 and 13 were inocculted

22-10-2013

Preparation of large plasmids

6 ml of clones pSB4K5+DelH B3, B9, B10, C4

Preparation of large plasmids

6 ml of clones ccdB 3, 7, 11, 13

Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/DelH_week26"