The purified DNA is visible as a clear band at the expected height (5th row), the vagueness of the band is due to the sample not being amplified. The negative control shows a smear, the positive control is not detectable.
<b> Figure 4. Agarose gel electrophoresis of amplified cDNA via PCR. (28/06/24)
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Indication of 4 rows (from left to right): gene ladder, negative control (using 2nd set of primers), positive control, cDNA with primers (using 1st set of primers). The 4th slot contains a very slight band indicating the amount of amplified DNA is very scarce. A slight band at around 7.5 kB can be seen. The amplification still results in big smears.
<b> Figure 5. Agarose gel electrophoresis of amplified cDNA via PCR. (01/07/2024)
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Very clear smears visible after PCR of F8 cDNA with another set of new primers and GFP with the standard GFP primers. 5th row is blank due to loss of the sample. In row 7 very vague bands are seen - low in the bottom of the gel and at the top. The size of GFP cDNA is 730 bp.
<b> Figure 9. Analysis of eGFP cDNA by nanodrop. (04/07/2024)
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An agarose gel electrophoresis was prepared to assess the purification and digestion of FVIII pcDNA plasmids (Figure 10). The digested plasmids displayed clear bands at the expected length of the vector – 12086 base pairs. [1] This attested the capability of these samples for further run-off IVT.
<b> Figure 10. Agarose gel analysis of digested F8 pcDNA plasmids. (08/07/2024)
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The A260/280 ratio detects protein contamination in samples. The A260/230 ratio detects salt contamination, data for samples H, I, J were lost during notebook keeping. The average concentration (ng/ul) is 2480 and 1847 for F8 and GFP, respectively. The average A260/280 ratio is 2.1 for both mRNA types. The average GFP A260/230 ratio is 2.5.
<b> Figure 13. Agarose gel analysis of transcribed samples via IVT. (29/08/2024)
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One of the IVT experiments did show promising results when analysing samples via nanodrop. The concentrations of both GFP and F8 were found to be high, the A260/280 and A260/230 ratio gave a good indication of purity. These ratios can estimate whether the sample is contaminated with protein structures and salts, respectively (Table 1). When analysing these samples on gel, no band presence was visualised. Possible cause thought to be mRNA splicing which would not have been detected on the 0.7 % agarose gel. All small RNA samples would have ‘run out’ during the electrophoresis process (Figure 15).
<b> Figure 21. Agarose gel electrophoresis of IVT samples. (09/07/24)</b> (To prevent waste, the same agarose gel is used for multiple experiments. The crossed out lanes are from a previous experiment.)
