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.
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
June 25th
Received the TdT del1-27. del26-143, and GIP subAAA variants!
Did a double digest of TdT variants and pSB1C3 ADD PROTOCOL
PCR purified restriction enzyme digest products and then performed a ligation with T4 DNA Ligase ADD PROTOCOL
June 26th
Performed a Transformation of TdT variants cloned into pSB1C3 with NEB5alpha cells
June 29th
Made two 1% agarose gels
Observed that colonies grew on our transformation plates
Did a colony PCR of multiple colonies
Ran PCR products on a gel
Made backup colonies for cells that had vector + insert
June 30th
Sent in pSB1C3 + TdT variant colonies to be sequenced
JULY 2015
July 1st
Followed Double Digest Protocol for Pet28b+ with BamHI and XbaI; this includes the double digest of Pet28b+ and TdT del 1-27. del 26-143, and GIP sub AAA TdT mutants
Did PCR purification - Really low concentration and bad graph on nanodrop
Was recommended to use less EB buffer in the future>
Reran digestion of Pet28b+
Did PCR purification with less EB buffer
Performed a ligation of Pet28b+ and TdT variants using T4 DNA Ligase
July 2nd
Did a transformation of ligations of Pet28b and TdT variants
July 6th
Performed inoculations of certain colonies
July 7h
Mini prepped inoculations from previous night
Nanodrop of colonies indicated good results and sent the samples to be sequenced
July 8th
Sequencing results indicated no priming for samples, and therefore ligation did not work or the DNA was lost in some process afterwards
July 9th
Re-ran double digests of Pet28b+ and TdT variants
Performed a gel purification of the digests and set up an overnight ligation of TdT variants + Pet28b+
July 10th
Made fresh LB Agar + Kanamycin plates
Transformed ligations from July 9th onto fresh Kan-plates
July 14th
Inoculated colonies of TdT variants + Pet28b+
July 15th
Did Colony PCR of TdT variants + Pet28b+ colonies
Ran a gel of colony PCR, but results came out negative
July 17th
Planned the immobilization of disulfide modified oligos to mercaptosilane treated glass slides
Designed a disulfide oligonucleotide for immobilization and ordered 3-mercaptopropyl trimethoxysilane
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
