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Team:University College London/LabBook/Week7
From 2012.igem.org
Contents |
Monday 23.7.12
Aim - Transformation of TetR BBa_C0040 BioBrick
Step 1 - Thawing cells: Thaw all materials on ice
Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes
| Component | Amount (ul) (one enzyme used) | Amount (ul) (two enzymes used) |
|---|---|---|
| dH20 | 2.5 | 1.5 |
| Buffer 1x | 1 | 1 |
| DNA template | 5 | 5 |
| BSA | 0.5 | 0.5 |
| Enzyme 1 | 1 | 2 |
| Enzyme 2 | N/A | 1 |
Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.
Step 4 - Centrifuge:
RPM: 14000
Time: 1 minute
Temperature: 18oC
Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:
RPM: 550
Time: 2.5 hours
Temperature: 37oC
Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:
RPM: 550
Time: 25 minutes
Temperature: 65oC
Monday 23.7.12
Aim - Repeat Restriction Digest for BioBricks in Expt 4.1 and 5.1, where the gel previously was inconclusive: This is intended to diagnose whether the correct plasmid had been transformed into our bacteria, by measuring the size of the digested products against a DNA ladder. In previous gel attempts, K540000 has produced a band of the correct size, but we are repeating it because of the presence of other unexpected bands, which we expect are contaminants from the reaction. A previous restriction digest of BBa_I13522 has failed to produce a band of the correct size, so we are repeating the digest before considering another transformation. For the same reason, we are repeating the digest of BBaK398108, which produced bands of incorrect size, which is suggestive of contamination.
(LOGO) Restriction Digest
Step 2: Set up as follows
| Samples | Recipe | Enzymes | |
|---|---|---|---|
| BioBricks | BBa_ K540000 (Expt 4.1) | Digested | Xbar 1 & Spe1 |
| BBa_ I13522 (Expt 4.1) | Digested | Xbar 1 & Spe1 | |
| BBa_ K398108 (Expt 5.1) | Digested | Xbar 1 & Spe1 | |
| BBa_ KI32003 (Expt 5.1) | Digested | Xbar 1 & Spe1 | |
(LOGO) Gel Electrophoresis
Results: The image below shows a 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 7.2. Visible in Lane 1 is a product of the correct size for the BBa_K540000 insert (3123bp), as indicated by A. Also shown is a correct sized product for the backbone PSB1C3 (2070bp), as indicated by B. Lane 2 shows a product corresponding to the size of the BBa_I13522 insert (937bp) as indicated by C. Also present is a product corresponding to the size of the PSB1A3 backbone (2155bp) as indicated by D. Lane 3 has a product corresponding to the expected size of insert BBa_K398108 (844bp) as indicated by E, and a product of the expected size for the plasmid backbone PSB1C3 (2070bp) as indicated by F. Lane 4 shows no products, where we would expect a product of size 1849bp (indicated by G) and a product of 2155 (indicated by H).
(Insert Picture 1)
Conclusion: Plasmids Bba_k540000 (3123), Bba_I13522 (937) and Bba_K391108 (844) have produced bands of the correct size. The band at 2000 is the plasmid backbone. The failure of Bba_K123003 to produce a band is not of great concern, as we do not expect we will require this BioBrick. However, we may repeat it at a later date.
Tuesday 24.7.12
Aim - Colony Picking from unsuccessful colonies for Expt 7.1 and 6.3
(LOGO) Picking Colonies
Step 2 – Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic required for each BioBrick.
| Samples | Number of Falcons | Broth | Antibiotic | |
|---|---|---|---|---|
| Biobrick | BBa_C0040 | 3 | Lysogeny Broth (5ml) | Ampicillin(50ug/ml) |
| BBa_I750016 | 4 | |||
| BBa_B0034 | 5 | |||
| BBa_B0015 | 3 | |||
| BBa_J23119 | 2 | |||
Wednesday 23.7.12
Aim - Results from Colony Picking
Results:
| Samples | Number of Falcons | Growth | |
|---|---|---|---|
| BioBrick | BBa_C0040 | 3 | None |
| BBa_I750016 | 4 | None | |
| BBa_B0034 | 5 | Growth in one | |
| BBa_B0015 | 3 | Growth | |
| BBa_J23119 | 2 | Growth | |
Conclusion: We are beginning to consider other possibilities for the failure of Colony Picking in Expt 7.1 and Expt 6.3. Foremost, we believe it may be due to the addition of Ampicillin to the Agar before it has cooled sufficiently, leading to degradation of the antibiotic. This would reduce the selective pressure, and allow growth of colonies that are not Ampicillin-resistant. Subsequent inoculation into Amipicillin positive LB brother leads to failed growth. Those BioBricks that have worked will undergo Miniprep, Restriction Enzyme Digests and Nanodrop.
Method
Miniprep: (LOGO) Miniprep Protocol 1 (Qiagen)
Step 1: Set up an eppendorf as follows
Step ?: Used 2mls
Step ?: Step ? had to be missed because we realised too late we had too little PE? buffer remaining.
Restriction Digest:
(LOGO) Restriction Digest Protocol 1
Step 2 - Setting up Digests and Controls: The protocol describes the recipe for (i) Digested Plasmid and (ii) Uncut Control. The table below indicates that an uncut and an Xcar1/Spe1 digested sample be set up for each BioBrick.
et up Eppendorfs as follows
| Samples | Recipe | Enzymes | |
|---|---|---|---|
| BioBrick | BBa_B0034 | Digested Plasmid | Xbar 1 & Spe1 |
| Undigested Plasmid (Control) | None | ||
| BBa_B0015 | Digested Plasmid | Xbar 1 & Spe1 | |
| Undigested Plasmid (Control) | None | ||
| BBa_B0034 | Digested Plasmid | Xbar 1 & Spe1 | |
| Undigested Plasmid (Control) | None | ||
(LOGO) Gel Electrophoresis
Results: The image below shows a 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 7.2. Visible in Lane 1 is a band corresponding to the Plasmid Backbone PSB1A2 (2079bp) as show by A, but it is not possible to detect our BBa_J23119 insert on this gel, as it is just 35bp long. Lane 2 shows the uncut plasmid for BBa_J23119 (shown by B) but the size difference between A and B appears to large for Bba_J23119 to have been the insert. This will require further investigation. Lane 3 shows a product 129bp long (indicated by C) which is the correct size for our insert BBa_B0015. A stronger band is also visible, indicated by D, which corresponds to the plasmid backbone PSB1AK3 (3189bp). Lane 4 displays a product which is somewhat larger than the expected size for the uncut plasmid (3318bp) as indicated by E. Lane 5 displays the correct product for the PSB1A2 backbone (2079bp) as indicated by F, but the extremely small size of the Bba_B0034 insert (12bp) means that it cannot be detect on this gel. Lane 6 indicates a band larger than expected, and does not correspond to the size of the uncut plasmid (2091bp) as shown by G.
(INSERT PIC 2)
Conclusion: The BBa_B0015 transformation was a success. However, with regard BBa_J23119, we feel that the difference in the product size of A (PSB1A2 plasmid backbone) and B (uncut plasmid) raises concerns as to whether the insert could be as short as 35bp. However, we must also consider the secondary effects had by the conformation of an uncut plasmid on its migration through the gel. It is possible this is sufficient to misplace the plasmid, such that its position does not represent its size. Even without this possibility, however, it would not be possible to determine whether there was a 35bp difference on such a crude scale of ladder. Instead we will reattempt the Analysis against a 25bp ladder, with the intention of detecting the insert. (See Expt 8.1). We have similar concerns for BBa_B0034, and so this too will be run against a 25bp ladder.
Nanodrop: (LOGO) Nanodrop Protocol 1 – Qiagen
| BioBrick | λ260 | λ 280 |
|---|---|---|
| BBa_J23119 (ng/μl) | 77.6 | 71.8 |
| BBa_B0034 (ng/μl) | 70 | 71.2 |
| BBa_B0015 (ng/μl) | 128.9 | 128 |
Week 7
