Team:UNIPV-Pavia/Calendar/October

101° Bio-Lab - Trasformed plates were chacked(I70, I71, I72 and I73).

Transformation of I55 into E. coli DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.

Miniprep and Nanodrop quantification for: I31, I35, I60 (they were also sent sequencing), INTEIN, pSB1C3-1, pSB1C3-2, pSB1C3-3 and I37 (they were no sent sequencing). Digest and gel run/cut/extraction/quantification of these samples were performed.

ON ligation of: I74: I31 + I35 and INTEIN_C3: INTEIN + pSB1C3.

102° Bio-Lab - PCR from colony for I55 (eleven colonies were picked).We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.

Miniprep and quantification for I73-1/2/3/4/5/6. 1,5 hours E-P digest (for screening) and gel run.

Transformation of I74 and INTEIN_C3 (1ul) in E. coli TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.

Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.

103° Bio-Lab - Glycerol stock for:I55-1, IC-1/2/3/4-4C5.

Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.

Miniprep and quantification of:I55-1, I55-1bis. I55-1bis was digested E-S for about three hours and than gel run/cut.

Colony PCR for I74-1..6. Gel run for PCR samples and I55 (E-S) and cut for this one.

Gel extraction and quantification of I55.

Ligation of I55 with already available DNA: I75: I55 + I37 and I77: I55 + I57.

Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.

104° Bio-Lab - Miniprep and quantification of: INTEIN_C3, I74-8, I54, I35. Digest and gel run/cut/quantification of these samples.

Ligations of I78=I74+INTEIN_C3.

Transormation (1ul) into E. coli TOP10 of:I75, I77. They were plated on LB+Amp agar plates and let grow ON at +37°C, 220 rpm.

105° Bio-Lab - Digestions were performed again for I55, I37, I57, I31 and I54. Digestions were let at +37°C ON.

Transformation (1ul) of I78 into E. coli TOP10. In order to check our E. coli TOP10 competent cells we plated them on LB+Amp and Cm agar plates.

ON ligation of I79: I60 + INTEIN_C3.

106° Bio-Lab - E. coli TOP10 plated on LB+Amp agar plate showed red colonies!!!

Transformation of available ligations I75 and I77 (1ul) into E. coli TOP10. They were plated on LB+Amp agar plates and let grow ON at +37°C, 220 rpm.

Gel run and cut for ON digestions and Nanodrop quantification of I54 and I55. Ligation of I76=I55+I54.

PCR from colony screening for I78.

107° Bio-Lab - I78 screening gel run. Glycerol stock and miniprep for I78-3.

Colony PCR for screening of I79 (we pick eleven colonies).

Transformation (1ul) of I0_1C3, I3_1C3, I7_1C3, I8_1C3, I9_1C3, I12_1C3 and I76 into E. coli DH5-alpha.

They were plated on LB+Cm34 agar plates (except for I76 that was plated on LB+Amp agar plate) and let grow ON at +37°C.

108° Bio-Lab - Glycerol stock, miniprep and Nanodrop quantification of I79-2.This DNA was sent to BMR Genomics for sequencing.

109° Bio-Lab -

110° Bio-Lab - Inoculum into 4 ml LB+Amp of: I75-1/2/3, I76-1/2, I77-1/2/3, I47, I48, I49, I63, I64 and I65.They were let grow at +37°C, 220 rpm.

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124° Bio-Lab -

125° Bio-Lab - Closing wiki