Showcasing Polysaccharide Production: 27th - 31st August 2012
**Tuesday 28/08/12**
9.30am
Using the NanoDrop Spectrophotometer the DNA concentrations of Becca Philp’s samples were checked for accuracy.
Table 1 Concentrations of DNA |
Sample |
ng/μl |
Cyclodextrin 1 |
90 |
Cyclodextrin 2 |
153 |
Hyaluronan Synthase (HAS) 1 |
66 |
Hyaluronan Synthase 2 |
92 |
SacB 1 |
106 |
SacB 2 |
94 |
Terminator 1 |
83 |
Terminator 2 |
86 |
10.00am
3A Assembly
Sample |
Conc of DNA (ng/μl) |
DNA required (μl) |
Water required (μl) |
Cyc 2 |
150 |
3.3 |
13.5 |
HAS 1 |
330 |
5.5 |
11.3 |
SacB 2 |
100 |
5.0 |
11.8 |
Upstream |
DNA |
500ng |
BUFFER (10x) |
2μl |
BSA (100x) |
0.2μl |
Water |
up to 20μl total V |
ENZYME 1 - ECORI |
0.5μL |
ENZYME 2 - SpeI |
0.5μL |
Downstream |
DNA |
500ng |
BUFFER (10x) |
2μl |
BSA (100x) |
0.2μl |
Water |
up to 20μl total V |
ENZYME 1 - XbaI |
0.5μL |
ENZYME 2 - PstI |
0.5μL |
Chloramphenicol Plasmid |
DNA |
500ng |
BUFFER (10x) |
2μl |
BSA (100x) |
0.2μl |
Water |
up to 20μl total V |
ENZYME 1 - ECORI |
0.5μL |
ENZYME 2 - PstI |
0.5μL |
11.00am
Incubated for 1 hour 37°C
12.00pm
Put samples into wells of 80°C to heat denature enzymes.
1.30pm
Created gel.
Ran gel electrophoresis, 150mV for ~20 minutes.
![](https://static.igem.org/mediawiki/2012/3/32/Exe2012_28-08-12_cyc_has_sacb.jpg)
Weighed eppendorf before and after addition of sliced gel portion containing DNA.
Re-separated DNA from gel
4.00pm
Ligation process.
Ligation |
Backbone |
2μl |
Upstream |
2μl |
Downstream |
2μl |
DNA ligase buffer |
1μl |
DNA ligase enzyme |
0.5μL |
Water |
2.5μL |
Plasmid + Upstream + Downstream
PSBC13 + Cyc2 + Term
PSBC13 + HAS1 +Term
PSBC13 + SacB2 + Term
Ligations left at 4°C overnight.
**Wednesday 29/08/12**
2.00pm
Transformations.
* pSB1C3 hyaluronan synthase
* pSB1C3 cyclodextrin + terminator
* pSB1C3 hyaluronan synthase + terminator
* pSB1C3 sacB + terminator
Equilibrated water bath to 42°C.
SOC medium kept at room temperature.
One Shot TOP10 competent cells, stored at -80°C.
2. Transferred 25μl to each eppendorf to be used.
3. Added 5μl of DNA (cyclodextrin, hyaluronan, and sacB)
2.15pm
4. Kept the tubes on ice for 30 minutes.
2.45pm
5. Heat shocked cells – transferred to water bath 42°C for 30 seconds exactly.
6. Moved straight back into the ice for ~2 minutes.
7. Aseptically added 125μl of room temperature SOC medium to each tube.
3.15pm
8. Incubated all samples in the shaker at 37°C, 250 rpm for 1 hour.
4.15pm
Span all samples at 3000G for 2 minutes.
Took 90μl off of each vial, this was then discarded.
Pipette mixed leftover contents of each eppendorf.
Using aseptic technique the rest was spread onto plates.
5.15pm
These were then put in an incubator overnight (~16+hours) at 37°C.
**Thursday 30/08/12**
9.30am
Checked plates. Colonies have formed on all.
These were then all placed in the fridge, 4°C.
4.30pm
Transferred cultures to liquid medium
Made up liquid broth using plates:
* pSB1C3 hyaluronan synthase
* pSB1C3 cyclodextrin + terminator
* pSB1C3 hyaluronan synthase + terminator
* pSB1C3 sacB + terminator
4x made per plate – aseptic technique
Used 10ml broth
10μl chloramphenicol
Scraped off single colony using pipette tip which is then ejected into liquid broth.
5.30pm
Put into horizontal shaker set at 37°C, 220rpm – left overnight.
**Friday 31/08/12**
9.30am
Mini-Prep
Liquid broth transferred to new containers – leaving pipette tip behind.
These were then centrifuged at 3900rpg for 10 minutes.
2. re-suspended pellet in 250μl re-suspension buffer, used up/down pipette mixing. Moved into eppendorf tubes.
3. added 250μl lysis buffer, mixed each by turning (upside down and back).
4. added 350μl neutralisation buffer.
5. centrifuged at full speed (13k) for 5 minutes. White build up had formed itself in the centre of the vials, centrifuged again for 5 minutes. Slight change, clear fluid became accessible.
6. clear fluid transferred to flow through tubes.
7. centrifuged for 1 minute --> flow through fluid discarded --> 500μl of wash solution added.
8. centrifuged for 1 minute --> flow through fluid discarded --> 500μl of wash solution added again.
9. centrifuged for 1 minute --> flow through fluid discarded --> centrifuged for 1 minute.
10. transferred column to clean eppendorf.
11. added 50μl clean water --> left for 2 minutes --> centrifuged for 2 minutes, kept vial, discarded column.
12.30am
Samples were then placed on the NanoDrop machine to get the concentration of DNA.
Using this and the measurements from the previous digestion, Table 2, the amount of DNA and water required for all samples could be calculated.
Table 2. Digest Measurements |
DNA |
500ng |
BUFFER (10x) |
2μl |
BSA (100x) |
0.2μl |
Water |
up to 20μl total V |
ENZYME 1 - ECORI |
0.5μL |
ENZYME 2 - PSTI |
0.5μL |
Amount of DNA and water required for all samples:
Sample |
Conc of DNA |
DNA required |
Water required |
CYCLODEXTRIN |
ng/μl |
μl |
μl |
1 |
40.7 |
2.29 |
4.51 |
2 |
55.8 |
8.93 |
7.90 |
3 |
41.1 |
12.2 |
4.6 |
4 |
947.5 |
0.53 |
16.3 |
HYALURONAN SYNTHASE |
ng/μl |
μl |
μl |
1 |
194.1 |
2.58 |
14.22 |
2 |
106.6 |
4.69 |
12.11 |
3 |
40.0 |
12.5 |
4.3 |
4 |
94155.4 |
3.22 |
13.58 |
CYCLODEXTRIN +TERMINATOR |
ng/μl |
μl |
μl |
1 |
100.4 |
5.0 |
11.8 |
2 |
142.8 |
3.5 |
13.3 |
3 |
16.3 |
30.7 |
1.0 |
4 |
110.8 |
4.5 |
12.3 |
HYALURONAN SYNTHASE + TERMINATOR |
ng/μl |
μl |
μl |
1 |
104.6 |
4.8 |
12 |
2 |
211.7 |
2.36 |
14.4 |
3 |
55.5 |
9.0 |
7.8 |
4 |
70.4 |
7.1 |
9.7 |
SacB + TERMINATOR |
ng/μl |
μl |
μl |
1 |
119.3 |
4.2 |
12.6 |
2 |
22.5 |
22.0 |
1.0 |
3 |
84.0 |
6.0 |
10.8 |
4 |
67.9 |
7.4 |
9.4 |
All samples were spun down.
2.45pm
Incubated all tubes for digestion period of 1 hour.
3.55pm
Samples out of incubator. 4μl of Loading Buffer added to each. These were then transferred to individual wells.
LADDDER – HAS+Term 1,2,3,4 – CYC+Term 1,2,3,4 – WbiP 2, 3, 4
Next row contains: LADDER – SacB+Term 1,2,3,4 – HAS 1,2,3,4 – CYC 1,2,4,