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.