Cooper Union iGEM 2015 Notebook
JUNE 2015
June 3rd - 8th
We designed various truncated and modified TdT sequences using the SnapGene viewer. The sequences were derived from the results documented in a paper by Repasky et. all detailing the Mutational Analysis of Terminal Deoxynucleotidyltransferase-mediated N-Nucleotide Addition in V(D)J Recombination.
The following sequences were designed: TdT delta1-27 (BBa_K1746000), TdT delta26-143 (BBa_K1746001), and TdT GIP 213-215 subAAA (BBa_K1746002).
June 16th
We practiced making 1% agarose gels. We immobilized DNA to Dynabeads (following the Dynabead Prep Protocol) and then ran experiments with TdT using regular dATPs and Cleanamp dATPs (following the Dynabead Cleanamp Protocol). We ran a gel of the experiments but saw no results. It is proposed that DNA was lost when removing DNA from Dynabeads.
June 17th
Prepared more Dynabeads with immobalized DNA (following the Dynabead Prep Protocol)
Prepared LB Broth
Prepared more 1% agarose gels
June 18th
Ran gels of Cleanamp Dynabead Experiment, but saw no results.
June 22nd
To test our polyacrimide gels (they were kind of old) we ran another polyacrimide gel with regular DNA sequences
High School Summer STEM Outreach.
We also spent our time perfecting our technique for creating mercaptosilane treated glass slides, and immobilizing disulfide modified DNA to them. This process involved a collaboration with the Genspace iGEM Team.
AUGUST 2015
August 17th
Set up another double digest of Pet28b+ and the TdT variants with XbaI and BamHI
August 18th
Performed a PCR cleanup of the double digests the Pet28b+ and TdT variants
Set up an overnight ligation of Pet28b+ and TdT variants
Set up an RNA ligase reaction of PNK treated 43mer and untreated 52mer
August 19th
To verify if ligation worked, instead of transforming immediately, performed PCR of ligation product
Ran an agarose gel of the PCR products, results were slightly conclusive; performed transformations of ligations of TdT variants + Pet28b+
Ran a polyacrimide gel of RNA ligation, and results were also inconclusive
August 20th
Performed colony PCR of 5 colonies from each plate (5 from TdT del1-27 + Pet28b+; 5 from TdT del26-143 + Pet28b+; 5 from TdT GIP subAAA + Pet28b+)
Results were positive! Indicated TdT del1-27 inserts in Pet28+
Made backup colonies of samples that had TdT del1-27 inserts
August 24th
Performed more colony PCR of the same plates from August 20th, to verify if any other colonies had inserts
Ran gel of colony PCR and verified cells with inserts containing TdT del26-143
Made backup colonies of samples that had TdT del26-143 inserts
Inoculated previous colonies containing TdT del1-27 inserts
August 25th
Mini-prepped inoculated TdT del1-27 colonies and sent plasmid to be sequenced
Inoculated previous colonies containing TdT del26-143 inserts
August 26th
Sequencing results verified Pet28b+ plasmids containing the TdT del1-27 inserts with an accuracies of over 97%!
Prepared and sent previous TdT del26-143 samples for sequencing
August 27th
Sequencing results verified Pet28b+ plasmids containing the TdT del26-143 inserts with an accuracies of over 98%!
Transformed all good TdT variants into Rosetta Protein Purification Cells
SEPTEMBER 2015
September 2nd
Inoculated Rosetta cells containing Pet28b+ with ligated TdT del1-27, and TdT del26-143
September 3rd
Used some inoculate to make backup colonies of Pet28b+ with ligated TdT del1-27, and TdT del26-143
Mini-prepped Pet28b+ with ligated TdT del1-27, and TdT del26-143
Designed and ordered a new reverse ligation oligo for RNA ligation reaction
September 8th
Set up RNA ligation with new reverse ligation oligo
