Melbourne/Lab Notebook Weeks 9-12
From 2007.igem.org
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Contents |
Week 9
19 Aug 2007
- Picked 3-4 transformants from "ligation" plates (17/8) and Grew up liquid cultures.
- Transformed the two ligation reactions in the pink rack in the cold room and plated on Gen + Amp plate.
20 Aug 2007
- Miniprepped liquid cultures from 19/8 labeled LA, LB, LC and P2 13K A, P2 13K B.
- Concentration of DNA was measured to be:
A- 221ng/ul
B-224ng/ul.
Desired products:
- 450bp (95bp different from CI-2 used as control)
- 450bp
- 450bp
- 1kb
- 1kb
- These were run on a gel.
- Digestions were set up for ligations.
OmpR-inverter
-Vector: OmpR (AmpR) miniprepped P1 15P from 10/7 digested with S/P.
-Insert: c1 inverter (1kb fragment) P2 13K A with X/P.
RBS-LacZ
-Vector: LacZ (KanR) P2 9E from 6/8 with E/X.
-Insert: RBS (GenR) P4 8J from 6/7 with E/S.
Digested for 2h and heat inactivated for 10min at 80degC.
- Made Glycerol Stocks of P2 13K using dry ice method.
21 Aug 2007
- Picked 4 colonies from fresh ligation/transformation to Growing up.
- These transformations were made as follows:
22 Aug 2007
- Miniprepped L1-L4 (liquid cultures from 21/8).
- Digested these for ligations.
- L1 X/P
- L2 X/P
- L3 X/P
- L4 X/P
- CI-2 X/P (control for 350bp)
- P1 15P S/P
- The above were Ran on a Gel together with P2 13K X/P digest from 21/8.
All L1-L4 had the 450bp desired band.
- Gel purified 450bp band from L3 and a 950bp band from the P2 13K digest.
- Ligated the following:
OmpR-L3 (RBS-LacZa-double term)
-Vector= P1 15P S/P (above)
-Insert= L3 X/P (above)
OmpR-c1 inverter (P2 13K)
-Vector= P1 15P S/P (above)
-Insert= P1 15P S/P (above)
Ligations were kept overnight at 4degC.
23 Aug 2007
- Transformed ligations from 22/8 into competent NM522 cells.
24 Aug 2007
- Liquid cultures were made of transformations from 23/8. 6 colonies were picked from each ligation.
25 Aug 2007
- Miniprepped cultures from 23/8 and M30109 cells.
- Digested:
- Ran on a Gel 16 samples that were miniprepped.
- Gel purified 1000bp fragment from ompR-c1 inverter ligation digest and a 600bp fragment from ompR-L3 ligation digest.
Week 10
26 Aug 2007
(all from 25/8) Left overnight at 4degC.
27 Aug 2007
Plated on Amp plates.
28 Aug 2007
- Picked six colonies from each plate (27/8) and cultured at 37degC for 13 hours.
29 Aug 2007
- Miniprepped 24 cultures from 29/8.
- Diagnosted all with X/S.
- Ran on a Gel
-The M30109 ligations yielded random fragments: something wrong with the part? - P1 15P-L3 and P1 15P-c1 inverter ligations had bands of the desired size.
30 Aug 2007
- Set up minipreps from 29/8 for sequencing.
OmpR-L3 and OmpR-c1 inverter were the desired sequences.
Parts created:
OmpR-RBS-LacZa-double terminator
31 Aug 2007
1 Sept 2007
Week 11
2 Sept 2007
- Digested and Ran on a Gel M30109 minipreps from 25/8 using a number of restriction enzymes to try to find out what it is:
- EcoRV
- Xho1
- EcoRV/Xho1
- E/P
- E/S
- E/X
- X/P
- X/S
- S/P
- E
- P
- S
- EcoR1/EcoRV/Xho1
- Apa1
- BamH1
- EcoR1/BamH1
The results showed that although a fragment of cph8 may be present in the part (1200bp fragment present only when double digested by EcoRV/Xho1), it may not be in the form as designated in the iGEM wiki.
The original miniprep was sent off for sequencing using vf2. The results showed that the part is a form of RBS-cph8, although the sequencing got cut off in the middle of the cp8 sequence. Therefore, the entire part designated is not present. We consequently decided to create the M30109 part ourselves.
3 Sept 2007
4 Sept 2007
- Made LB (600ml) + LB-agarose (400ml)
- Created Agar Plates with Kan.
5 Sept 2007
- Recieved ComP and ComA sequences from GeneArt.
6 Sept 2007
- Transformed the following on Amp plates except where labeled
7 Sept 2007
- Liquid cultured a colony from each of the 6/9 transformations. No growth was observed for the M30109 undigested plates and were discarded.
8 Sept 2007
- Miniprepped cultures from 7/9.
- Digested these with X/P
- Ran these on a Gel.
-All had desired sizes.
- Glycerol Stocks of all four of these were created.
Week 12
9 Sept 2007
- Ligated the following:
ComP-backbone vector
-Vector: P3 20I Death Plasmid AmpR X/P fragment from 8/9.
-Insert: ComP X/P fragment from 8/9.
ComA-backbone vector
-Vector: P3 20I Death Plasmid AmpR X/P fragment from 8/9.
-Insert: ComA X/P fragment from 8/9.
10 Sept 2007
11 Sept 2007
12 Sept 2007
- Transformed ligations from 9/9.
- Also Transformed resuspended DNA for the following:
- [http://partsregistry.org/Part:BBa_I15008 P2 21A] (ho1)
- [http://partsregistry.org/Part:BBa_I15009 P2 21A] (PcyA)
- [http://partsregistry.org/Part:BBa_J61034 P4 8J] (IPTG sensory device)
- [http://partsregistry.org/Part:BBa_R0040 P1 7O] (tetR negative promoter)
- [http://partsregistry.org/Part:BBa_B0015 P1 1I] (Double terminator)
and minipreprs from previous dates:
- Efficiency of fast ligations were simultaneously tested for. Using 2X ligase buffer, 2hour and 5min ligations were tested using ComA parts from 8/9. These did not have ligation efficiencies as high as the overnight ligations but were sufficient. The 5 min ligation seemed to have more colonies than the 2 hour ligation.
13 Sept 2007
- P4 8J and P1 5G were overgrown so were streaked onto new plates.
- Colony PCR using The Endy Procol [http://openwetware.org/wiki/Endy:Colony_PCR] of 2 colonies from ComA-cell death (12/9) and OmpR-L3 (27/8),
- 5ul 10 X PCR buffer (green)
- 5ul MgCl2 25mM
- 0.5ul vf2
- 0.5ul vR
- 1.0ul dNTPs 25mM
- 0.5ul GoTaq
- 37.5ul milliQ water
on a cycle of:
- 94C for 5min
- 94C for 30 seconds
- 55C for 30 seconds
- 72C for 1:15 minutes
- repeat 2-4 for 30 times
- 72C for 7min.
14 Sept 2007
15 Sept 2007
