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*[[Hosuk:Lab_Notes|LabNote]] ===Rolony, 11th : Result=== *Done at [[Hosuk:Lab_Notes/2013-06-23|06/23]] ====Result==== *'''SUCCESS!!''' *Number of rolonies are much more than previous experiments and their density is so high. *EM gain need to be decreased because of so much high contrast. *10uL Phi29 looked more brighter than 3uL sample. **EM gain for 10uL is 5, and 10 for 3uL. *But 3uL of Phi29 is enough. *Summary of the differences from previous before going to Boston **Adjust each concentration of Riboshredder+RNase H+RNase H Buffer (Jonathan's protocol) **Use original CircLigaseII master mix (2uL of enzyme, no doubling other reagents), 4hr CircLigase reaction **Enzymatics phi29, high concentration (I used 1uL previously and I tried 3uL and 10uL this time, for testing) **pre-annealing RCA primer in 2x SSC+30% formamide **and this time I used new vial of aminoallyl-dUTP, I though previous one was almost running out and too old. ====Next Plan==== *Compare between… *# Epicentre vs. Enzymatics Phi29 *# 1uL vs. 3uL Phi29 *# 2hr. vs. 4hr. CircLigase II *# Strip and re-hybridize Cy5 probe ====Epi, 20x, NA=0.8, S3(3uL Phi29)==== *Raw images : [[Media:S3_3uLPhi29Enzymat_20x-NA0.8_pos02_Cy3_exp0.1-EM10.tif|Cy3]], [[Media:S3_3uLPhi29Enzymat_20x-NA0.8_pos02_BF_exp0.1-EM00.tif|BF]] [[File:S3_3uLPhi29Enzymat_20x-NA0.8_pos02.png|500px]] ====Epi, 20x, NA=0.8, S4(10uL Phi29)==== *Raw images : [[Media:S4_10uLPhi29Enzymat_20x-NA0.8_pos03_Cy3_exp0.1-EM5.tif|Cy3]], [[Media:S4_10uLPhi29Enzymat_20x-NA0.8_pos03_BF_exp0.1-EM00.tif|BF]] [[File:S4_10uLPhi29Enzymat_20x-NA0.8_pos03.png|500px]]
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