We first kept all the reagents ready before proceeding with the experiment.
We were in short of Sodium acrylate and did not order as we didnt plan to do the experiment.When I looked for alternatives for Sodium acrylate, I found that disposable diapers have this chemical, we took it out and removed the cotton stuck to the diaper, took just the sodium acrylate, minced together and observed that it expanded when mixed in water. We took 2 solutions and planned to standardize the same, as we did not get 38% solution. Images of preparation of sodium acrylate is as shown below:
We had Acrylamide and Bis acrylamide solution at the ratio of 28:2. The other reagents were added as mentioned in the protocol.
We tweaked the protocol in order to standardize the procedure for getting the gel expanded. The standardization procedure is as mentioned below:
| Reagents | Monomer Solution 1 (100ul) | Monomer Solution 2 (100ul) | Monomer Solution 3 (100ul) | Monomer Solution 4 (400ul) |
|---|---|---|---|---|
| Acrylamide - Bisacrylamide (28:2) | 13.75ul | 13.75ul | 13.75ul | 55ul |
| 4M Sodium Chloride | 40ul | 40ul | 40ul | 160ul |
| 10x Phosphate Buffer Saline | 10ul | 10ul | 10ul | 40ul |
| Water | Didnt add water separately | Didnt add water separately | Didnt add water separately | 141 ul |
| Sodium Acrylate | 12ul from 1% Sodium Acrylate solution(dissolved in water) | 12 ul from 0.5% Sodium Acrylate solution (dissolved in water) | 12 ul from 2% Sodium Acrylate solution (dissolved in water) | Minced 1g of Sodium acrylate vortexed without water |
| APS | 2ul | 1ul | 0.5ul | 2ul |
| TEMED | 2ul | 1ul | 0.5ul | 2ul |
Why did we change the concentration? |
Sodium acrylate was already dissolved in water, so water was not added.APS and TEMED same concentration as given in the protocol |
Sodium acrylate was already dissolved in water, so water was not added.APS and TEMED concentration was reduced as it polymerized immediately even before we could pour the monomer solution. |
Sodium acrylate was already dissolved in water, so water was not added.APS and TEMED concentration was reduced further as it polymerized in less than a minute. |
Water was added separately and the sodium acrylate that was minced was added to the monomer solution was vortexed before adding APS and TEMED.APS and TEMED concetration was increased as the volume of the solution increased. |
We did not observe any expansion of gel in the first 3 monomer solutions
We observed that the monomer solution 4 expanded in 1 hour and 2 hours. This is probably due to the fact that in the first 3 gels, we added the sodium acrylate already mixed with water as solution, and the sodium acrylate has already expanded in water. But when we tried adding only the minced sodium acrylate salt into the monomer solution directly and vortexed the solution, we observed that the gel expanded.
Exercise 2: Expansion of antibody-stained E. coli
Fluorescent In Situ Hybridization of expanded bacterial genomes Prepare the FISH probes using nick translation
We might not be able to do this experiment here due to the lack of FISH probes in our facility.
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