Team:Stockholm/15 October 2010

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====Results====
====Results====
Results indicate no significant difference in growth between different cultures. Slightly lower curve for the nTra10-expressing clone, which is somewhat interesting, since a similar progress was seen in the canceled experiment from 14/10. At least one more experiment will be run next week, using new BL21 clones; this will show whether this is just a coincidence or not.
Results indicate no significant difference in growth between different cultures. Slightly lower curve for the nTra10-expressing clone, which is somewhat interesting, since a similar progress was seen in the canceled experiment from 14/10. At least one more experiment will be run next week, using new BL21 clones; this will show whether this is just a coincidence or not.
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== clone non-CPP operon ===
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==== colony PCR ====
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 +
{|
 +
! mix
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|
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| x16
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| rowspan="5" width="150" |
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! primers
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| rowspan="5" width="150" |
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! samples
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|-
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| mastermix
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| 24.5
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|
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| pSB_VF2
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| pSB1C3.SOD.RBS.yCCS 1-5
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|-
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| DNA
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| 0.5
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|
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| pSB_VR
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| pSB1C3.SOD.his.RBS.yCCS 1-5
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|-
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| align="right" | tot
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| 25µl
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|
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| rowspan="2" |
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| pSB1C3.his.SOD.RBS.yCCS 1-5
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|-
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| colspan="3" |
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| positive control (pSB1C3.LMWP.SOD.his)
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|}
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==== Gel ====
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[[Image:2010-10-15 pC.S yC.jpg|200px|thumb|left|]]
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{|
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! well
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! sample
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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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| 12
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| ladder
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|-
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| 2
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| pC.S_yC 1
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| 13
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| pC.hS_yC 1
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|-
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| 3
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| pC.S_yC 2
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| 14
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| pC.hS_yC 2
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|-
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| 4
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| pC.S_yC 3
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| 15
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| pC.hS_yC 3
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|-
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| 5
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| pC.S_yC 4
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| 16
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| pC.hS_yC 4
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|-
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| 6
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| pC.S_yC 5
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| 17
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| pC.hS_yC 5
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|-
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| 7
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| pC.Sh_yC 1
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| 18
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| pC.L.hS, control
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|-
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| 8
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| pC.Sh_yC 2
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| rowspan="4" |
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| rowspan="4" |
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|-
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| 9
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| pC.Sh_yC 3
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|-
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| 10
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| pC.Sh_yC 4
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|-
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| 11
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| pC.Sh_yC 5
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|}
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==== ON culture ====
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3ml LB Cm<sub>25</sub> + toothpick from colony
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SOD.RBS.yCCS 5
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SOD.his.RBS.yCCS 2
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his.SOD.RBA.yCCS 3
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 +
{{Stockholm/Footer}}

Latest revision as of 11:10, 26 October 2010

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Contents

Nina

Continuation of protein purification

Washing

I washed the column with 10 X volumes of wash buffer.

The Ni-resin (in the form of pellet) is 10 ml, therefore 10 * 10ml Ni-NTA = 100 ml wash buffer.

The imidazole is 2 M.

2M * Volume = 100 * 30 *10^-3 M Volume imidaziole that needs to be added in the wash 100 ml is 1.5 ml

Washing:

Aq14.jpg

Elution

I eluted the column with 5 X volumes of elution buffer.

The Ni-resin (in the form of pellet) is 10 ml, therefore 5 * 10ml Ni-NTA = 50 ml elution buffer.

  • 20 ml 40 min on shake 4°C
  • 10 ml 30 min on shake 4°C
  • 10 ml 30 min on shake 4°C
  • 10 ml 30 min on shake 4°C

I saved these samples in the cold room 4°C for tomorrow.

Agarose gel verification

I ran the PCR products on an 1 % agarose gel 80 V to check if I had any inserts.

Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp

Aq12.jpg

Arrangement on gel:

Aq13.jpg

Gels:

Aq15.jpg

Unfortunately non of the bands show that there has been an insert, it might be that the transformation into BL21 cells was not a good idea since they are not cloning cells, but we are short on time and I tried to make a short cut. It might have failed this time. I will run a new screen with 16 colonies/dish, it that won't work then this cloning has not worked out well.

Andreas

Growth curve assay

Setup

Inoculated new flasks from ON cultures set 14/10:

  • 250 ml E-flasks
  • 25 ml LB + Amp 100 + 1 mM IPTG
  • 250 μl ON culture
    1. BL21 pEX.SOD⋅His
    2. BL21 pEX.nTra10⋅SOD⋅His
    3. BL21 pEX.nTAT⋅SOD⋅His
    4. BL21 pEX.nLMWP⋅SOD⋅His
  • 37 °C, 225 rpm

OD590 measurements made every 30 min with appropriate dilution in fresh LB.

OD590 measurements

  0 min 30 min 60 min 90 min 120 min 150 min 180 min 210 min 240 min 270 min 300 min 330 min 360 min 390 min
pEX.SOD⋅His 0.035 0.047 0.090 0.180 0.195 0.176 0.251 0.177 0.239 0.140 0.164 0.205 0.196 0.193
pEX.nTra10⋅SOD⋅His 0.037 0.050 0.092 0.163 0.156 0.132 0.175 0.130 0.164 0.116 0.155 0.167 0.154 0.168
pEX.nTAT⋅SOD⋅His 0.033 0.046 0.090 0.170 0.177 0.161 0.225 0.153 0.225 0.045 0.170 0.162 0.193 0.200
pEX.nLMWP⋅SOD⋅His 0.031 0.042 0.86 0.174 0.192 0.178 0.237 0.170 0.242 0.137 0.180 0.185 0.200 0.200
Dilution factor 1 1 1 1 2 4 5 10 10 20 20 20 20 20
Line chart of plotted OD(590) measurements from growth assay.

Results

Results indicate no significant difference in growth between different cultures. Slightly lower curve for the nTra10-expressing clone, which is somewhat interesting, since a similar progress was seen in the canceled experiment from 14/10. At least one more experiment will be run next week, using new BL21 clones; this will show whether this is just a coincidence or not.


Mimmi

clone non-CPP operon

colony PCR

mix x16 primers samples
mastermix 24.5 pSB_VF2 pSB1C3.SOD.RBS.yCCS 1-5
DNA 0.5 pSB_VR pSB1C3.SOD.his.RBS.yCCS 1-5
tot 25µl pSB1C3.his.SOD.RBS.yCCS 1-5
positive control (pSB1C3.LMWP.SOD.his)

Gel

2010-10-15 pC.S yC.jpg
well sample well sample
1 ladder 12 ladder
2 pC.S_yC 1 13 pC.hS_yC 1
3 pC.S_yC 2 14 pC.hS_yC 2
4 pC.S_yC 3 15 pC.hS_yC 3
5 pC.S_yC 4 16 pC.hS_yC 4
6 pC.S_yC 5 17 pC.hS_yC 5
7 pC.Sh_yC 1 18 pC.L.hS, control
8 pC.Sh_yC 2
9 pC.Sh_yC 3
10 pC.Sh_yC 4
11 pC.Sh_yC 5


ON culture

3ml LB Cm25 + toothpick from colony

SOD.RBS.yCCS 5 SOD.his.RBS.yCCS 2 his.SOD.RBA.yCCS 3





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

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