Team:University College London/LabBook/Week8

Monday 30.6.12

Aim: Repeat the gel from Expt 7.3 (week 7) with a smaller ladder and higher agarose percentage in order to detect the very small inserts of BBa_J23119 (35bp) and BBa_B0034 (12hp). Previous ladder used was 10037 bp, and this time we used a 25bp ladder.

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 Xba1/Spe1 digested sample be set up for each BioBrick. Set up Eppendorfs as follows

Results:The image below shows a 3.5% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 7.3. The high agarose concentration of the gel was intended to slow the progression of DNA fragments to enable us to detect the small inserts of BBa_J23119 and BBa_B0034. The table below indicates the expected sizes of BioBricks and Plasmid Backbones. Accordingly, we used a the smallest available ladder, with fragments ranging from 25bp to 500bp. At the top of the gel it is possible to see the large fragments we detected on a 1000bp gel in Expt 7.3 Week 7. However, we had expected this gel to also demonstrate the BBa_J23119 insert (35bp) corresponding to letter A in Lane 1, and the BBa_B0034 insert, corresponding to letter B in Lane 3. No product was found. As expected, there was no product measurable against the 25bp ladder for the uncut plasmids in Lane 2 and Lane 4.

Conclusion:No product was detected for either BioBrick insert. However, we do no feel this necessarily proves the transformation failed, especially as we detected the correct plasmid backbone for each in week 7 (Expt 7.3). The modest nanodrop concentration obtained last week suggests that the concentration was not high enough to allow such a small fragment to be visualised. We are considering alternatives such as running a polyacrylamide gel, which is more sensitive to small fragments, or amplifying the fragment using PCR

Monday 30.7.12

Aim - Picking colonies: Colonies were picked for BBa_I750016, which demonstrated colony formation in Expt 7.4 on Friday 27.7.12.

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.

Tuesday 31.7.12

Aim – Results from Colony Picking

Results: The table below indicates whether there was growth for BBa_I1750016

Samples		Volume Inoculated	Colony Formation
BioBrick	BBa_I1750016	10μl	Yes
		90μl	Yes Conclusion: We can proceed onto Miniprep, Analytical Digest, and Nanodrop. Method Miniprep: Miniprep Protocol 1 (ANACHEM) Step 1 - Pellet Cells: Pellet 1.5-5ml bacterial culture containing the plasmid by centrifugation g = 6000 Time = 2 min Temperature = (15-25oC) Step 2 - Resuspend Cells: Add 250ul S1 to the pellet and resuspend the cells completely by vortexing or pipetting. Transfer the suspension to a clean 1.5ml microcentrifuge tube. Step 3 - Puncturing Cell Membrane: Add 250ul S2 gently mix by inverting the tube 4-6 times to obtain a clear lysate. Incubate on ice or at room temperature for NOT longer than 5 min. Step 4 - Neutralising S2: Add 400ul Buffer NB and gently mix by inverting the tube 6-10 times, until a white precipitate forms. Step 5 - Centrifuge: g: 14000 Time:10 minutes Temperature: (15-25oC) Step 6 - Centrifuge: Transfer the supernatant into a column assembled in a clean collection tube (provided. Centrifuge: g = 10,000 Time: 1 minute Temperature: (15-25oC) Step 7 - Wash Column: Discard the flow-through and wash the spin column by adding 700ul of Wash Buffer. Centrifuge: g - 10,000 Time - 1 minute Temperature: (15-25oC) Step 8 - Remove residual ethanol: Centrifuge: g - 10,000 Time - 1 minute Temperature: (15-25oC) Step 9 - Elute DNA: Place the column into a clean microcentrifuge tube. Add 50-100ul of Elution Buffer, TE buffer or sterile water directly onto column membrane and stand for 1 min. Centrifuge: g - 10,000 Time - 1 minute Temperature: (15-25oC) Step 10 - Storage: Store DNA at 4oC or -20oC Restriction Digest: Restriction Enzyme Digest Protocol 1 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 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 Xba1/Spe1 digested sample be set up for each BioBrick. Set up Eppendorfs as follows Samples Recipe Enzymes BioBrick BBa_I150016 Digested Plasmid Xba1 & Spe1 Undigested Plasmid (Control) None 8-1 8-2 8-3 8-4 8-5