Progress Report- 1027

Binding assay data here

Cloning and sequencing started here

After a small scale clone check, a large scale amplification of 12 colonies per plate was performed and it was ascertained that from Round 2, all of the clones were sequenced, from Round 7A, clone 1 was omitted due to ambiguity of purity of sequences, from Round 7B, clones 2, 6, 8, 9 were omitted due to failure of amplification, and from Round 10B, everything was sequenced.

Following sequencing, in Round 2, it was complete randomness of sequences. In 7A, there was also randomness in the sequences, but in 7B, clones 1, 7, and 12 were almost identical in sequence, leading to the assumption that this particular sequence should have a higher binding affinity for the protein 1027. There was then an attempt to assemble the sequences together to establish similarities.

These are the three sequences from R7B that were identical. The primer regions are identified by the green arrows, while the space inbetween identifies the random oligonucleotide that is used for selection and binding to the target protein.

In round 7A, we were unable to find most of the primer regions for both the reverse and forward pool primers. The sequences were not purified enough perhaps, but we were unable to tell what the sequences of the oligonucleotides were.

In Round 10, there were primer dimer formations that are both bad and inexplicable, other than an annealing temperature that was too low at the time of amplification.

As shown, the primers line up against one another creating artifacts that amplify faster than other actual sequences. This shows an assembly of 7 of the 12 clones from round 10B that were sequenced. Unfortunately, artifacts cannot be removed, so if selection were to be resumed, it would have to be resumed at a round before 10.

The clones from R7B and R7A were grown up in LB + Amp broth overnight and then regrown to plasmid prep for cleaned plasmids with inserts. Unfortunately, after the second day, only 1, 10, and 11 grew in addition to 7 from R7A. They were plasmid prepped and sequenced with primers added by the sequencing core.

As shown in the image above, once again only amplification artifacts were shown. Troubleshooting for the cause of this is still in process.

After this, a second cloning event was performed. The ligation and cloning were performed per invitrogen and TA cloning manual instructions, respectively. These techniques were first introduced in Spring 2010. The cloning yielded both blue and white colonies as shown below:

The numbered colonies were the white ones selected for further analysis. The clone check for colonies 1, 7, 14 and 24 are shown below:

All but clone 14 worked, which was enough to do amplification on all of the clones and do a largescale clone check, which will be done later. Clone 1 from the previous cloning will be amplified and sequenced again.

Things to continue doing: Ligating sequences from R7B into plasmids and cloning them to establish a complete library of sequences in R7B and analyze for similarities that may aid in binding affinity, sequencing the plasmids from the clones, analyzing the sequences and possibly assembling together the clones with similar sequences.


Brad Hall said...

Can you repost these pictures, they aren't showing up...again.

Katherine Li said...

The pictures are showing up when I open the page.