Amplification of sgRNA-Cas9 constructs and results for restriction digestion of repair template

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Lane 1: 5ul of 2 log 1.0-10KB NEB ladder

•Lane 2: TN: PCR + RE digest. (N-terminal fusion). Tube code for PCR reaction? Tube code for restriction enzyme digest reaction? Which plasmid was digested? Which enzymes were used? 

•Lane 3: RV: I mixed the PCR reaction RV 19/16 and RE digest RV RE 10 together. The PCR reaction includes the template, pJW1219 (isolate: MG030416_10), forward primer, MG 2211 and reverse primer MG2203. Bands are not very clear but adequate number of colonies grew following SDM and transformation. The digest reaction includes pDD 286 digested with Avr II and NgoM IV. No bands were seen RE Digest might not have worked.

Line 4: (labels on gel are incorrect) diagestion reaction product NK-17. Repair template pDD282GFP was digested with restriction enzymes ClaI and SpeI.There're two band on gel in 6500 and 2600 bp range, which means that digestion reaction worked.

Line 5: PCR product NK-19, include 4ul of PCR and 1ul of dye. Touchdown PCR product was amplified using MG030416-10 template, Q5 hot start 2x Master mix, 2212 and 2203 primers. Band is not very bright, but is i right range (6500bp), it means that PCR reaction was successful.

Lane 6: 

Lane 7:

Authors: Mo Kaze, Sean Collier and Alex Hermes

Lane 1: 2-log ladder (0.10-10Kb) from NEB

Lane 2 - KZ: touchdown PCR reaction for isolate MG030416_10 (PCR code: KZ13) with Forward Primer MG2208 and Reverse primer MG2214. No results appeared for this lane and subsequent colonies did not grow when this product was used for a Site Directed Mutageneis KLD reaction and E. coli transformation.

Lane 3 - KZ: Digestion reaction for pDD 286 RFP N-terminal fusion with restriction enzymes ClaI and NgoMIV (Digest code: KZ11). Bands appear around the 6500bp, 2600bp and 610bp regions. These bands appear as expected.

Lane 4 - AH9: Digested plasmid pDD282-1 (GFP) with 2 restriction enzymes (which restriction enzymes?). We see 3 expected bands of 6598, 2635, and 631 bp. 

Lane 5 - AH11: PCR amplification for unc-11 C-Terminal fusion, project 5. Template ID: 040316-10. Performed PCR amplification with 2 primers: MG2203_sgRNA_R_6141, MG2212_Unc-11_C. . No PCR bands seen for product but achieved successful transformation.

Lane 6 - SC

Lane 7 - SC