Team:UCL/Labbook/Week15

From 2013.igem.org

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These ligation reactions were stored at -20 degrees Celsius and used in the following day for transformation.
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These ligation reactions were stored at -20 degrees Celsius and used in the following day for <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformation</a>.
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These were incubated overnight and then minipreped.
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These were incubated overnight and then <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a>.
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MMP9 in cmv hygro plasmid was used to transform home-made competenet cells in order to built a stock.  
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MMP9 in cmv hygro plasmid was used to transform home-made <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> competent cells</a> in order to built a stock.  
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Using the cells of a remaining cell vial of homemade competent cells, 10 LB agar plates were spread
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Using the cells of a remaining cell vial of homemade competent cells, 10 <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> LB agar plates</a> were spread
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We transformed 7 homemade competent cells using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.
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We transformed 7 homemade <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> competent cells</a> using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.
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After the transformation procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.
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After the <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformation</a> procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.
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Re-inoculations of glycerol stocks of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone.   
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Re-inoculations of <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a> of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone.   
There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.
There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.
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  - For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp
  - For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp
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Prep digest of samples from minipreped inoculations 1, 14, 16, 17, 24.
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Prep digest of samples from <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a> inoculations 1, 14, 16, 17, 24.
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Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific <a href="http://tools.lifetechnologies.com/content/sfs/manuals/oneshottop10_man.pdf" target="_blank"> transformation protocol</a> for this type of cells was followed apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant.
Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific <a href="http://tools.lifetechnologies.com/content/sfs/manuals/oneshottop10_man.pdf" target="_blank"> transformation protocol</a> for this type of cells was followed apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant.
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3 LB agar plates with 5x chloramphenicol were prepared and the 3 transformations were spread on these.
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3 LB agar plates with 5x chloramphenicol were prepared and the 3 <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformations</a> were spread on these.
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Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band).  
Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band).  
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Prepared 9 glycerol stocks from the 9 inoculations made the day before while the rest of each of these inoculations was minipreped.
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Prepared 9 <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a> from the 9 inoculations made the day before while the rest of each of these inoculations was <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a>.
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Latest revision as of 01:33, 5 October 2013

Lab Weeks

Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18

Week 15

Bacterial Labs

Monday 9th September

Contamination control was set using 15 ml Falcons with 3 ml LB broth and 3 ul cmp or 3 ul amp.

Retransformation of zeocin pSB1C3 ligations from 4 September and 6:1 zeocin and cmv ligations from 31 of August.

Tubes ligations used: 5z, 6z, 7z, 8z and 9, 10 controls from 4 of September and 2z from 31 of August. These were plated and left over-night.

LIGATION 3:

pSB1C3 digested and purified, concentration = 25 ng/ul

zeo digested and purified conc = 77 ng/ul

CMV digested and purified conc = 25 ng/ul

Zeocin (77 ng/ul) 3 to 1 (mass ratio) [z1] 6 to 1 (mass ratio) [z2]
water (ul) 6 4
Quick ligase buffer (ul) 10 10
backbone (ul) 2 2
insert (ul) 2 4
ligase (ul) 1 1
Total 21 21

Cmv (25 ng/ul) 3 to 1 (mass ratio) [c3] 6 to 1 (mass ratio) [c4]
water (ul) 2 3
Quick ligase buffer (ul) 10 10
backbone (ul) 2 1
insert (ul) 6 6
ligase (ul) 1 1
Total 21 21

Controls 5 ctrl check no circular backbone 6 ctrl check digestion process 7 ctrl (uncut backbone)
water (ul) 7 18 18
Quick ligase buffer (ul) 10 0 0
backbone (ul) 3 3 3 of uncut backbone
insert (ul) 0 0 0
ligase (ul) 1 0 0
Total 21 21 21

These ligation reactions were stored at -20 degrees Celsius and used in the following day for transformation.

Reinoculated K812014 from glycerol stocks in order to increase the stocks of backbone pSB1C3. 15 ml Falcon contained 3 ul cell culture, 3 ml LB broth and 3 ul chloramphenicol (cmp).

These were incubated overnight and then minipreped.

Tuesday 10th September

Colony counts of the re-transformation of ligations:

Plate Colony counts
2z from 31/08 100
5z from 4/09 150+
6z from 4/09 80+
7z from 4/09 100
8z from 4/09 0
9z - ctrl from 4/09 40
11z - ctrl from 4/09 0

MMP9 in cmv hygro plasmid was used to transform home-made competent cells in order to built a stock.

Colony count - 90% - 100+ and 10% 80 colonies.

Biobricks (K81 and J63) as well as potential recombinant plasmid transformations (transformations of 31 August) were inoculated from glycerol stocks and minipreped in order to achieve a strong stock of pSB1C3 backbone.

The minipreps resulted showed an average concentration of about 30 ng/ul (highest concentrations were found for one of the miniprep of K812014 biobrick - 34.6 ng/ul and for a transformation of cell vial 28: 67.6 ng/ul.

The following day, some of these, showing high concentrations, were E and P analytical digested in 30 ul reaction volumes. The nanodrop result of the MMP9 miniprep was 42 ng/ul with 260/280 = 2.05.

Building up the competent cell stock using the home-made competent cells

Using the cells of a remaining cell vial of homemade competent cells, 10 LB agar plates were spread

Plate number Drug Volume competent cells (ul) Colony count
I 1000 X CMP 20 60
II 500 X CMP 20 15
III 250 X CMP 20 0
IV N/A 20 100+
V 1000 X CMP 20 10
VI 500 X CMP 20 3
VII 250 X CMP 20 0
VIII N/A 20 100+
IX 1000 X CMP 100 from T vial 15
X N/A 100 from T vial 100+
I 1000 X CMP 20 60

Colonies from plate IX and X were picked and taken through the competent cell test using 250 x (4x) for drug plate.

New inoculations (in 3 ml LB broth and 3 ul cmp) of the transformations from 4/09 and 9/09 (?) using 8 colonies from each of the following plates: 2 z, 5 z, 6 z, 7 z were prepared and left in the incu-shaker in the usual conditions over night.

Transformation number 3:

We transformed 7 homemade competent cells using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.

The c3 ligation (1:3) was lost as one of the cell vials containing was taken by mistake by someone from the team.

After the transformation procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.

On the same day, 10 ul of z2, c4 and pSecTag2A (of potentially transformed cells) was inoculated with 2 ul cmp and 2 ml LB broth.

Wednesday 11th Sepetmber

Re-inoculations of glycerol stocks of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone. There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.

Labeling of the 8 colony inoculations of each of the potential transformation with potentially recombinant pSB1C3 + zeocin (31 and 4/09 transformations):

Falcon Tube no. Content: Plate & Colony ng/ul 260/280
1 2z.1 54.0 1.82
2 2z.2 28.3 2.21
3 2z.3 25.6 3.03
4 2z.4 20.3 3.16
5 2z.5 13.4 3.08
6 2z.6 15.7 2.61
7 2z.7 8.9 14.17
8 2z.8 14.8 4.08
9 5z.1 64.9 1.70
10 5z.2 36.5 1.93
11 5z.3 16.6 2.01
12 5z.4 32.1 2.22
13 5z.5 23.6 2.50
14 5z.6 53.3 1.87
15 5z.7 33.7 2.22
16 5z.8 50.1 2.06
17 6z.1 74.8 1.71
18 6z.2 32.6 1.82
19 6z.3 26.0 2.25
20 6z.4 42.5 2.15
21 6z.5 30.8 2.02
22 6z.6 27.3 2.23
23 6z.7 38.0 2.15
24 6z.8 43.5 2.10
25 7z.1 62.7 1.80
26 7z.2 31.7 1.89
27 7z.3 26.4 1.88
28 7z.4 21.4 2.12
29 7z.5 13.1 2.28
30 7z.6 27.2 2.22
31 7z.7 - -
32 7z.8 33.0 2.28

Colony counts of the transformed cells with the ligation prepared on the 9/09

Plate Colony Count
Z1 12
Z2 0
C4 1 (+small colonies)
5 50
6 50
7 17
pSecTag2A 10

Inoculations for Z1 and C4 were prepared (3 colonies for Z1 plate were picked as well as the only colony on C4 plate; all inoculatons were made in 2 LB broth and 4 ul chloramphenicol and left for 16 hours in the incu-shaker at 200 rpm, 370 C).

Prep digest of linearised pSB1C3 from the 2013 High school distribution kit with EcoR1 and Pst1 in a 40 ul volume reaction (using 25 ul DNA and 2 ul of each E and P). This was incubated for 2 hours and then freezed. Before it was used in another ligation, it was heat inactivated at 80 degrees Celsius for 20 min).

Analytical digest of zeocin and K812014 biobrick with Stu1 restriction enzyme. Expected bands: - For zeocin 2 bands: one of 1037 bp and another of 783 bp - For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp

Item ul
DNA zeocin (55 ng/ul)/K812014 (34.6 ng/ul) 5
Stu1 1
Buffer 4 1
dH2O 3
Total 10

The zeocin tube (PCR purified) of 77 ng/ul was diluted by adding 10 ul of RO water (new concentration around 55 ng/ul). These cuts were run on a gel, next to uncuts.

Thursday 12th September

Prep digest of samples from minipreped inoculations 1, 14, 16, 17, 24.

Contents Volumes (ul)
DNA 14
Stu1 0.5
Buffer 2 10
BSA 0.5
dH2O 5
Total 30

After EcoR1 and Pst1 digestion and running a gel of the potential backbone sources for building up the pSB1C3 stocks we decided to avoid using biobrick K812014 due to the presence of 3 bands instead of 2. Instead we decided to use J632014 biobrick as a pSB1C3 source for now on because it shows the correct number of bands and lengths of the digested DNA material.

Prep digest of pSB1C3 (E, P digested the day before) with Dpn1 – added 2 ul of Dpn1 to the volume of 40 ul of double digested pSB1C3 and incubated it for 1 hour at 37O C. This was done in order to prepare the backbone for ligation. Also, before ligation, the tube was heat inactivated at 80O C for 22 minutes.

Ligation 4

Ligations AA1 BB1 CC1
EP cut, purified pAZEC fragment (53ng/ul) 2 0 2
*EPD cut pSB1C3 2 2 0
T4 DNA ligase buffer 1 1 1
**Vol. of dilute T4 ligase 1 1 1
RO Water 4 6 6
Total 10 10 10
Cell Counts 0 0 0

* before EPD (E –EcoR1, P- Pst1, D-Dpn1) digestion it was in a concentration of 25 ng/ul;

** Diluted by adding 4 ul of RO water to 4 ul of DNA T4 ligase from Alex. These were left on the bench for 30 minutes and then heat inactivated for 20 minutes at 80O C.

Transformation 4

Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific transformation protocol for this type of cells was followed apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant. 3 LB agar plates with 5x chloramphenicol were prepared and the 3 transformations were spread on these.

Friday 13th September

PCR of bb zeocin using Taq PCR kit New England Biolabs in 50 ul reaction volume.

Nanodrop readings of the miniprep of the inoculations of Z1 from the 11/09

Sample ng/ul 260/280
Z1.1 24.8 1.80
Z1.2 35.4 1.98
Z1.3 34.2 1.93

Accomplished tasks asked by Darren:

- Spread straight from the glycerol stock of J632014 on 4 plates (0 x cmp, 2 x cmp, 4 x cmp and 5 x cmp) in order to build up the stocks of pSB1C3 backbone source. There was no growth the following morning.

- Spread the remainder of transformed cells (12/09): 50 % on 5 x cmp plate while the other half on 2 x cmp. These were pelleted for 4 minutes.

- Plan MMP9 PCR with Taq DNA Polymerase: 2 reactions making use of the 2 types of primers as well as running 2 controls (with no template).

- New transformation using One Shot Top10 Competent Cells. For each ligation type, spread the cells 50% on 1xcmp and the other half on 5xcmp.

Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band).

Contents E&P digest (ul) BscR1 digest (ul) Stu1 digest (ul)
DNA 5 5 5
EcoR1 1 0 0
Pst1 1 0 0
Stu1 0 0 1
BscR1 0 1 0
Buffer 3 1 0 1
Buffer 4 0 1 0
BSA 0.5 0.5 0.5
RO H2O 1.5 2.5 2.5
Total 10 10 10

Saturday 14th September

Colony counts of the transformation prepared on 12th and 13th of September.

Plate AA1 5xcmp AA1 2xcmp AA1 1xcmp BB1 5xcmp BB1 2xcmp BB1 1xcmp CC1 5xcmp CC1 2xcmp CC1 1xcmp
12th Sep 1 0 - 3 28 - 0 0 -
13th Sep 0 - 30 0 - 3 0 - 0

8 inoculations in 2 ml LB and 1xcmp were made using picked colonies from plate AA1, 1xcmp. 1 inoculation was prepared in 4xcmp for the singular colony on AA1 5 x cmp which grew after more than 20 hours of incubation.

Nanodrop result of these minipreps – data from the next day

AA1 1xcmp colony number ng/ul 260/280
1 26.8 1.86
2 24.8 1.94
3 35.1 1.90
4 28.2 1.95
5 30.5 1.94
6 28.7 1.99
7 35.1 1.85
8 40.3 1.88

The inoculation in 4xcmp from the plate AA1 5xcmp showed a concentration of 40.1 ng/ul and 260/280 coefficient equal to 1.89.

PCR of bb zeocin was set using Taq polymerase to make three 50 ul volume reactions and a control with no pSecTag2A template.

A gel was run to check MMP9 PCR and zeocin PCR from 5/09 samples z1, z2, z5, z7 and control 9. The correct bands appeared apart from z2 and z7 which showed no DNA.

Sunday 15th September

Prepared 9 glycerol stocks from the 9 inoculations made the day before while the rest of each of these inoculations was minipreped.

From the plates of the day before, other 20 colonies from AA1 1xcmp plate (falcons 1.1 – 1.20) and 3 colonies from AA1 5xcmp (falcons 5.1 to 5.3) were picked and inoculated in 4xcmp LB broth.

Ligation and transformation of One Shot Top ten Competent cells with MMP9 and EPD triple digested pSB1C3 (form high school iGEM distribution kit – label hs):

Volume (ul) MMP9 4bb MMP9 4bb Control (no backbone) Control (no insert) MMP9 3
Water 2.5 2.5 5 7.5 2.5
pSB1C3 2.5 (hs) 2.5 (from K812014) 0 2.5 (hs) 2.5 (hs)
T4 ligase buffer 10 10 10 10 10
T4 ligase 1 1 1 1 1
Insert - MMP9 5 5 5 0 5
Total 21 21 21 21 21
Vial Labels 1 2 3 4 5

Mammalian Labs

Tuesday 10th September

Threw away 12 over-confluent ( aprox. 150%) plates (P15). 5 P16 plates with 90% confluency were passaged and then split 1:4. Made up 5 new p17 plates.

Added FBS and PS intp DMEM media to make new solution.

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