Linearized pdCas9-w is expected to be 6705 bp.
We tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. Many, but not all, of our samples were successfully amplified (cf. Fig.1).
For next steps, sample from lane 1 (cf. Fig.1) was used.

Successful PCR reactions are expected to yield 340 bp fragments.
PCR was succesful for sample visible on gel. (cf. Fig.2)

Amplicons are 666 bp if Gibson assembly worked and 396 bp if the plasmid self-ligated.
Lane "C" is a negative control: PCR was run with all components except template DNA. It is empty which means there is no contamination.
Gibson assembly seems to have worked for some samples. (cf. Fig.3)
To avoid working with too many samples, we kept the ones from lanes 16, 22, 25, 31, 37 and 43 for the next steps. We did overnight liquid cultures of these colonies.

pdCas9-w samples from different colonies are present on gels in triplicates in the following order:

• Undigested - expected to yield 7 kb circular plasmid (migrates faster than linear fragments of the same size)
• Digested by BamHI - expected to yield two fragments of 6147 bp and 834 bp if insert is present or one 6705 bp fragment if it is not
• Digested by KpnI - expected to yield two fragments of 45334 bp and 2447 bp if insert is present or one 6705 bp fragment if it is not

BamHI and KpnI are both unique cutters in pdCas9 (without the insert) and double cutters in pdCas9-w (with the insert).
Too much ladder was loaded so it is difficult to estimate the size of the fragments. The smaller fragments are also very difficult to see.
By looking at the relative heights, we can say that all colonies seem to have the w subunit insert. There is only the undigested sample for colony 16 that is not visible, probably due to an error while loading the gel.
Sequencing confirmed that sample 22 is in fact pdCas9-w. We used this sample for the next steps and stored it as a glycerol stock (c.f. Protocols).

The digested and undigested plasmids should have the same size. Circular fragments migrates faster than linear fragments of the same size. This means that the undigested plasmid should migrate faster than the digested plasmid.
Even though the ladder is not clear, by looking at the relative heights we can see that the digested sample migrated more slowly than the undigested sample (cf. Fig.9). The restriction digest was successful.

Note that this was repeated several times since we needed a lot of linearized pdCas9-w.

All amplified sgRNA cassettes are expected to be about 370 bp.

sgRNA cassette Z0 was amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-BtoD and primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Q5 PCR. Both samples were successfully amplified (cf. Fig.10a).

sgRNA cassette Z4 was amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. Samples 3 and 4 were successfuly amplified (cf. Fig.10b).

sgRNA cassette Z35 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. However, it is not easily visible on the gel. (cf. Fig.10c)

sgRNA cassette X0 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10d)

sgRNA cassette X4 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10e)

sgRNA cassette X35 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10f)

In colony PCR, primers were placed such as amplicons are 445 bp if the plasmid self-ligated, 723 bp if Gibson assembly of 1 sgRNA cassette worked and 1120 bp if Gibson assembly of 2 sgRNA cassettes worked.

pdCas9-w-Z0 was obtained by a faulty assembly of pdCas9-w-Z0-Z4 that only took up 1 of the 2 sgRNA cassettes. Sample 20 of the colony PCR (cf. Fig.11a) was confirmed by sequencing to be pdCas9-w-Z0 and to not contain any mutations that may have an effect on its activity. Sample 13 seems to be pdCas9-w-Z0-Z4 according to colony PCR (cf. Fig.11a), but sequencing showed that it is pdCas9-w-X0-Z0, probably due to contamination of a tube. (This sample will be used again in Part 2.)
Colony PCR showed that all colonies tested for pdCas9-w-Z4 have the inserted sgRNA cassette (cf. Fig.11b). Sequencing comfirmed that sample 5 is in fact pdCas9-w-Z4. Colony PCR showed that the Gibson assembly for sample 5 of pdCas9-w-Z35 seems to have worked (cf. Fig.11c) and sequencing confirmed that is the case. Sequencing also showed some mutations or deletions in the promoter and/or terminator for these 2 samples. We decided to keep working with these and test whether these mutations are significative with an activity assay.
Colony PCR and sequencing showed that Gibson assembly was successful for sample 5 of pdCas9-w-X0 (cf. Fig.11d). However, it contains a mutation in the promoter. A 2nd pdCas9-w-X0 without mutations will be constructed in part 2, after which we will be able to compare the activity of this promoter with/without a mutation.
Colony PCR of pdCas9-w-X4 showed many potentially good samples (cf. Fig.11e). Sequencing of sample 10 confirmed that it is in fact pdCas9-w-X4 and that it does not contain any mutations.
Colony PCR and sequencing showed that Gibson assembly was successful for the colony of pdCas9-w-X35 (cf. Fig.11d) and no mutations were found in the sequence.

Successful PCR reactions are expected to yield 4400 bp amplicons.
Both negative control lanes (cf. Fig.13) are empty, which means there is no contamination. Phusion PCR did not work, but Q5 PCR did (cf. Fig.13). We will work with this sample for next steps.

Successful PCR reactions are expected to yield 340 bp amplicons.
Both negative control lanes (cf. Fig.14) are empty, which means there is no contamination. Both Phusion and Q5 PCR worked (cf. Fig.13). We will work with the sample amplified by Q5 PCR.