1154 pCAMBIA-2301-Ara-s1

Cloning of Arabidopsis S1 Nuclease into pCAMBIA-2301

Purpose: Cloning of Arabidopsis S1 Nuclease into pCAMBIA-2301

Date:  05/24/2012

Notebook: 

Page: 008803-003308

Clone ID: 1154

Experimenters:  Jennifer Truong

Cloning

Date: 05/24/2012

PCR Template:

Isolated band for cloning next week

Missing template gel

Colony Screening

PCR Screening showed 100% efficency, grow samples 1-6 (lanes) in medium

Prepared Miniprep for samples 1-6, digested incorrect enzymes BglII and Nhe1

Submitted 1154-4 for sequencing, yielded by sequencing results, redid digest with Xho1 and Nco1

Digest successfully shows insert in all samples, submitted 1154-1 for sequencing

6/4/2012: 1154 turned on mutated, submitted 1154-5 for sequencing.

Result intepretation/Summary

Summary:

Oligomix 1: 1 uL of 10uM 1-14

Oligomix 2: 1 uL of 10mM 13-26

Reaction mixture: 0.5uL of oligomix 0.25uL Kapa HiFi polymerase

5uL 10x PCR Buffer

1uL of 10mM dNTP

18.25uL of water

PCR at Kapa setting 10 cycles with 30 seconds extension

Combined both reaction mixtures after PCR , add 1uL of 10mM Ara-S1-1 and 1uL of 10mM Ara-S1-26. PCR setting 25 cycles with 30 seconds extension.

Added 10.0 uL of 6x dye to PCR sample, load ~15 uL of sample into each well (lane one and two), loaded 5 uL of 1 kB buffer, ran gel in 0.5x buffer 15 minutes.

Conclusion

No results because the PCR lid was not on tight enough, solution evaporated, compromising the Mg composition of the solution resulting in all of the DNA to not be digested and remained in the well.

5/30/2012: After redoing the PCR, was able to succesfully transform the cells that were grown on the media.