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

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

July 20th-August 17th

During this time period, we spent most of our time working on our 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