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Lane 1: 5ul of 2 log 1.0-10KB NEB ladder
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.
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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.
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ReplyDeleteLine 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.
ReplyDeleteLine 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 2: TN-11 which is the combination of TN-7 and TN-9. 5ul of TN-7 mixed with 4ul of TN-9 and 1ul blue juice in the tube for a total of 10ul into the gel well. TN-7 was the product of enzyme digestion. DNA template 040316 measured by NanoDrop of 386.6ng/ul, and it was cut by ClaI and SpeI enzymes. TN-9 was the product of exponential amplification. MG030416 DNA template (measured by NanoDrop showed the concetrantion od 717/5ng/ul) added with MG2211 forward primer and MG2203 reverse primer.
ReplyDeleteLane 2 shows two unclear bands. The upper band predicted to be 6598 bp and the lower band could consist of 2635 bp. This indicates the enzyme digestion worked although the products are difficult to observe on the gel.
ReplyDeleteLane 6: Enzymatic digestion reaction of repair template pDD286RFP (MA-5) performed using restriction enzymes ClaI and NgoMIV. Estimated band sizes are 6500 and 2600 bp range.
ReplyDeleteLine 7: Touchdown PCR product (MA-6) consisting of MG030416-10 (template), 2212 (fwd) and 2203 primers (rev). Based on specification of NEB Q5 hot start high-fidelity.**