Performed plasmid isolation on 17 bacterial cultures Emma inoculated yesterday using the QIAprep Spin Mini Kit (Qiagen) using ~1.4mL of culture according to the manufacturer’s protocol. Plasmid DNA was eluted with 50uL of Buffer EB. Tubes were stored @ 4C in the refrigerator in FTR 213.

Selected 10 colonies (1-8, 18, 28) for mini-preps. Inoculated 5mL 1x LB + 50ug/mL of Kanamycin. Incubated O/N, 37C, 200RPM. 3mL of each culture were used for mini-preps. Used Qiagen kit. Samples were eluted w/30uL of EB.

Isolated plasmid DNA from 3mL of liquid cultures that were inoculated yesterday using Qiagen’s miniprep kit. DNA was eluted with 50uL of EB. DNA was prepped and sent for sequencing to ASU sequencing facility. Each clone was sequenced two times in each direction. Samples are as follows:

Name – Clone # Primer

• SJW01 – 1 M13F
• SJW02 – 1 M13F
• SJW03 – 1 M13R
• SJW04 – 1 M13R
• SJW05 – 2 M13F
• SJW06 – 2 M13F
• SJW07 – 2 M13R
• SJW08 – 2 M13R
• SJW09 – 3 M13F
• SJW10 – 3 M13F
• SJW11 – 3 M13R
• SJW12 – 3 M13R
• SJW13 – 4 M13F
• SJW14 – 4 M13F
• SJW15 – 4 M13R
• SJW16 – 4 M13R

Clone #s are as follows:

1 – 5′ Library Top band

2 – 5′ Library Mid band

3 – 5′ Library Bottom band

4 – 3′ Library band

Results:

Sequencing results received 20110801. SJW15 and 16 apparently stop abruptly. The sequencing facility believes this to be caused by secondary structure of the template. Depending on how things align, I may consider using 7-daeza-GTP in a PCR reaction and re-sequencing this clone, as the 7-daeza-GTP helps relax secondary structure.

Spoke with Steven and he suggested just designing new primers closer to each other and resubmit.

Plasmid was isolated from 3mL of liquid culture started yesterday using the Qiagen MiniPrep Kit, according to the manufacturer’s protocol. Will send off for sequencing.

Mini prepped 3mLs of each culture, according to Qiagen protocol. Samples were eluted with 50uL of Buffer EB.

• #9
• #10
• #13
• #43
• #45
• #49
• #56
• #58