Team:Stockholm/26 August 2010

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Procedures according to the standard colony PCR protocol. Elongation time 0:45.
Procedures according to the standard colony PCR protocol. Elongation time 0:45.
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== Mimmi ==
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=== His.SOD/His.yCCS ===
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==== plasmid mini rep ====
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*Transfer cell culture  to two 12ml falcon tubes
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*Spinn down the cells at 3000rpm for 10m
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*Follow the E.Z.N.A mini prep kit 1 protocol
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**Wash two times with DNA Wash buffer
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**Eluate in 50µl sH<sub>2</sub>O
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:::'''DNA concentration'''
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:::{| border="0"
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! His.SOD
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! His.yCCS
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|-
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| ~160ng/µl
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| ~230ng/µl
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|}
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=== MITF-M ===
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==== Gel ====
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[[Image:2010-08-26_pSB1C3.MITF-M.jpg|200px|thumb|left|]]
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{|
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! well
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! sample
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|-
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| 1
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| ladder
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|-
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| 2
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| MITF 1
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|-
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| 3
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| MITF 2
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|-
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| 4
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| Positive control
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|}
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==== New transformation ====
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*Follow the original protocol!
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*Let the plate dry in the incubator before use
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=== pEX.RFP ===
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==== verification PCR ====
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{|
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! Mix
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| (µl)
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| X5
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| rowspan="8" width="80" |
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! Primer
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| rowspan="8" width="150" |
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! colspan="2" | Conditions
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| rowspan="3" |
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|-
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| sH<sub>2</sub>O
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| 22.5
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| 112.5
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| pEX_VF
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! time
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! &deg;C
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|-
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| F primer
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| 1
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| 5
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| pEX_VR
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| 2m
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| 94
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|-
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| R primer
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| 1
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| 5
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| rowspan="5" |
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| 30s
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| 94
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| )
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|-
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| DNA
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| 0.5
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| 5X0.5
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| 30s
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| 60
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| > 30 cycles
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|-
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| align="right" | tot
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| 25µl
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|
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| 2m
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| 72
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| )
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|-
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| rowspan="2" colspan="3" |
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| 10m
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| 72
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| rowspan="2" |
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|-
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| oo
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| 10
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|}
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=== CPPn ===
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==== quick transformation ====
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*Keep on ice 5m
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*Heat shock in 42&deg;C for 30s
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*Cool down on ice
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*Plate on Amp-plate (dried in the incbator)
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{{Stockholm/Footer}}

Latest revision as of 11:00, 26 October 2010

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Contents


Hassan

[http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000347190%250D9606.ENSP00000270142%250D9606.ENSP00000368424%250D9606.ENSP00000353940%250D9606.ENSP00000295600%250D9606.ENSP00000241052%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000267082%250D9606.ENSP00000281043%250D9606.ENSP00000343052%250D9606.ENSP00000364094%250D9606.ENSP00000257880&channel1=off&channel2=off&channel3=off&channel4=off&channel5=off&channel6=on&channel7=on&custom_limit=0&interactive=yes&network_flavor=actions&required_score=700&targetmode=proteins] [http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000347190%250D9606.ENSP00000270142%250D9606.ENSP00000368424%250D9606.ENSP00000353940%250D9606.ENSP00000295600%250D9606.ENSP00000241052%250D9606.ENSP00000360157%250D9606.ENSP00000302777%250D9606.ENSP00000373571%250D9606.ENSP00000354130%250D9606.ENSP00000370171%250D9606.ENSP00000267082%250D9606.ENSP00000281043%250D9606.ENSP00000343052%250D9606.ENSP00000364094%250D9606.ENSP00000257880&channel1=off&channel2=off&channel3=off&channel4=off&channel5=on&channel6=on&channel7=on&custom_limit=0&interactive=yes&network_flavor=actions&required_score=700&targetmode=proteins]

Andreas

Assembly of SOD/yCCS⋅His constructs into pSB1K3

Sequencing results

From 23/8 BLAST results of

pSB1K3.SOD⋅His 1 (pK-SH1)
pSB1K3.SOD⋅His 2 (pK-SH2)
pSB1K3.yCCS⋅His 2 (pK-yH2)
pSB1K3.yCCS⋅His 3 (pK-yH3)

sequences revealed that no His tag was present. In fact, although SOD and yCCS were digested with AgeI for the assembly, the insertion found between SpeI and PstI had not been removed, indicating that SOD and yCCS hadn't even been properly digested; this makes it even more puzzling how the picked clones have been to grow on Km 50 plates, as the SOD and yCCS source plasmid was pSB1C3.

The only reasonable explanation is that SOD and yCCS have been digested only by EcoRI. After enzyme inactivation, however, PstI (which cannot be heat-inactivated) has digested the genes, making them complementary for insertion into pSB1K3 target vector.

Troubleshooting

Two controls were prepared to identify the problems with SOD/yCCS⋅His cloning:

  1. Gel verification of all samples digested with AgeI and EcoRI
    • Dig. pSB1K3.yCCS E+A (yCCS)
    • Dig. pSB1K3.SOD E+A (SOD)
    • Dig. pMA.His E+A (pMA)
  2. Plating of sequenced clones onto both Cm 25, Km 50 and Amp 100 plates, to verify the vector resistance.
    • pSB1K3.SOD⋅His 1
    • pSB1K3.SOD⋅His 2
    • pSB1K3.His⋅SOD 1
    • pSB1K3.His⋅SOD 3
Gel verification
Gel verification of samples digested with EcoRI and AgeI.
1 kb λ = O'GeneRuler 1 kb DNA ladder.
50 bp λ = GeneRuler 50 bp DNA ladder.

1 % agarose, 110 V, 40 min

Results
Fairly poor digestion of yCCS and SOD. Unclear for pMA.His, since the His fragment is too small to appear on the gel.

Transfer of RFP cassette into pEX

Transformation results

From 25/8

Good yield of both red and white colonies. Four red clones picked for colony PCR verification. PCR performed by Mimmi.

Tranfer of MITF into pSB1C3

Continued from 25/8

MITF taken over by Mimmi.

Amplification of N-CPPs

PCRs prepared from N-CPP cluster plasmid to isolate our three N-CPPs.

  • N-Tra10
    • Fwd: pSB-VF2
    • Rev: pSB-VR
  • N-TAT
    • Fwd: pEXf
    • Rev: pEXr
  • N-LMWP
    • Fwd: pGexf
    • Rev: pGexr

Procedures according to the standard colony PCR protocol. Elongation time 0:45.



Mimmi

His.SOD/His.yCCS

plasmid mini rep

  • Transfer cell culture to two 12ml falcon tubes
  • Spinn down the cells at 3000rpm for 10m
  • Follow the E.Z.N.A mini prep kit 1 protocol
    • Wash two times with DNA Wash buffer
    • Eluate in 50µl sH2O


DNA concentration
His.SOD His.yCCS
~160ng/µl ~230ng/µl


MITF-M

Gel

2010-08-26 pSB1C3.MITF-M.jpg
well sample
1 ladder
2 MITF 1
3 MITF 2
4 Positive control




New transformation

  • Follow the original protocol!
  • Let the plate dry in the incubator before use



pEX.RFP

verification PCR

Mix (µl) X5 Primer Conditions
sH2O 22.5 112.5 pEX_VF time °C
F primer 1 5 pEX_VR 2m 94
R primer 1 5 30s 94 )
DNA 0.5 5X0.5 30s 60 > 30 cycles
tot 25µl 2m 72 )
10m 72
oo 10


CPPn

quick transformation

  • Keep on ice 5m
  • Heat shock in 42°C for 30s
  • Cool down on ice
  • Plate on Amp-plate (dried in the incbator)





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/26_August_2010"