Team:Stockholm/31 July 2010

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(New page: {{Stockholm/Top2}} ==Nina== ==Coomassie gel on IPTG treated IgG protease== I removed the coomassie solution from both the IgG protease gel and also the bFGF gel. Then I poured a couple o...)
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==Nina==
==Nina==
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==Coomassie gel on IPTG treated IgG protease==
+
===Coomassie gel on IPTG treated IgG protease===
I removed the coomassie solution from both the IgG protease gel and also the bFGF gel. Then I poured a couple of mls of destaining solution onto the gels and left ON.
I removed the coomassie solution from both the IgG protease gel and also the bFGF gel. Then I poured a couple of mls of destaining solution onto the gels and left ON.
 +
 +
==Andreas==
 +
 +
===LMWP ligation/construction===
 +
''Continued from 30/7''
 +
 +
====Transformation results====
 +
 +
Colonies on all four plates, however only one colony on three of them. These were also not discovered until after colony PCR was prepared. Interestingly, the plate with many colonies was the only construct carried on the pSB1A3 vector:
 +
 +
:pSB1C3.N-LMWP 1:10 = 1 colony
 +
:pSB1C3.N-LMWP 1:100 = 1 colony
 +
:pSB1C3.C-LMWP 1:10 = 1 colony
 +
:pSB1A3.C-LMWP 1:100 = many colonies
 +
 +
====Colony PCR====
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 +
6 clones (A, B, C, D, E, F) were picked from the "pSB1A3.C-LMWP 1:100" plate for colony PCR:
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 +
=====Colony PCR (illustra ready-to-go PCR beads)=====
 +
*22.5 &mu;l dH<sub>2</sub>O
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*1.0 &mu;l VF2
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*1.0 &mu;l VR
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*0.5 &mu;l cell suspension
 +
 +
Positive control: pSB1A3.BBa_J04450 purified plasmid.
 +
 +
'''Amplification'''
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#95 &deg;C - 10:00
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#95 &deg;C - 0:30
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#55 &deg;C - 0:30
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#72 &deg;C - 1:00
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#*Cycle back to step 2 29 times.
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#72 &deg;C - 10:00
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 +
=====Gel verification=====
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[[Image:LMWP_colPCR_31jul.png|200px|thumb|right|Gel verification of LMWP colony PCR samples.<br>'''Loading:''' &lambda; - A - B - C - D - E - F - Pos contr. - Blank - Lig. N-LMWP&dagger; - Lig. C-LMWP&dagger;<br>&dagger; ''Samples of the 1:100 ligation mixtures from 30/7''<br><br>&lambda;=GeneRuler 50 bp DNA ladder.
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]]
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1 % agarose, 90 V, 60 min
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 +
Loaded volume:
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*GeneRuler 50 bp DNA ladder: 2 &mu;l
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*Samples: 4 &mu;l
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''Results''
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From what the results show, I chose the wrong ladder. Still, bands are unreasonably large (1500 bp ?). Interesting band at &asymp;300 bp in sample A. Since the pSB1A3 vector itself gives about 270 bp from the colony PCR, the band is probably too small to carry anything of interest.
 +
 +
===Plasmid prep===
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''Continued from 30/7''
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From ON cultures yCCS A & B, MITF A-D, MITF RED A & B.
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{|border="1" cellspacing="0" cellpadding="2"
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|'''Sample'''
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|'''Conc [ng/&mu;l]'''
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|A<sub>260</sub>/A<sub>280</sub>
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|-
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|pSB1C3.yCCS A
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|94.77
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|1.86
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|-
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|pSB1C3.yCCS B
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|85.78
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|1.93
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|}
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 +
DNA conc. not measured for MITF samples, as these will only be used for PCR.
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===Glycerol stocks===
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''Continued from 30/7''
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From ON cultures of yCCS A & B.
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 +
Old non-mutagenized glycerol stocks of yCCS were discarded.
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{{Stockholm/Footer}}

Latest revision as of 10:57, 26 October 2010

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Contents

Nina

Coomassie gel on IPTG treated IgG protease

I removed the coomassie solution from both the IgG protease gel and also the bFGF gel. Then I poured a couple of mls of destaining solution onto the gels and left ON.

Andreas

LMWP ligation/construction

Continued from 30/7

Transformation results

Colonies on all four plates, however only one colony on three of them. These were also not discovered until after colony PCR was prepared. Interestingly, the plate with many colonies was the only construct carried on the pSB1A3 vector:

pSB1C3.N-LMWP 1:10 = 1 colony
pSB1C3.N-LMWP 1:100 = 1 colony
pSB1C3.C-LMWP 1:10 = 1 colony
pSB1A3.C-LMWP 1:100 = many colonies

Colony PCR

6 clones (A, B, C, D, E, F) were picked from the "pSB1A3.C-LMWP 1:100" plate for colony PCR:

Colony PCR (illustra ready-to-go PCR beads)
  • 22.5 μl dH2O
  • 1.0 μl VF2
  • 1.0 μl VR
  • 0.5 μl cell suspension

Positive control: pSB1A3.BBa_J04450 purified plasmid.

Amplification

  1. 95 °C - 10:00
  2. 95 °C - 0:30
  3. 55 °C - 0:30
  4. 72 °C - 1:00
    • Cycle back to step 2 29 times.
  5. 72 °C - 10:00
Gel verification
Gel verification of LMWP colony PCR samples.
Loading: λ - A - B - C - D - E - F - Pos contr. - Blank - Lig. N-LMWP† - Lig. C-LMWP†
† Samples of the 1:100 ligation mixtures from 30/7

λ=GeneRuler 50 bp DNA ladder.

1 % agarose, 90 V, 60 min

Loaded volume:

  • GeneRuler 50 bp DNA ladder: 2 μl
  • Samples: 4 μl

Results From what the results show, I chose the wrong ladder. Still, bands are unreasonably large (1500 bp ?). Interesting band at ≈300 bp in sample A. Since the pSB1A3 vector itself gives about 270 bp from the colony PCR, the band is probably too small to carry anything of interest.

Plasmid prep

Continued from 30/7

From ON cultures yCCS A & B, MITF A-D, MITF RED A & B.

Sample Conc [ng/μl] A260/A280
pSB1C3.yCCS A 94.77 1.86
pSB1C3.yCCS B 85.78 1.93

DNA conc. not measured for MITF samples, as these will only be used for PCR.

Glycerol stocks

Continued from 30/7

From ON cultures of yCCS A & B.

Old non-mutagenized glycerol stocks of yCCS were discarded.





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

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