Dinh/Dinh 2014/NOTES/2014-6-23
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Stoffel production[edit]
Stoffel plasmid construction[edit]
- Re-purify pOpenTaq
2 x 5 mL overnight cultures in 50 ug/mL ampicillin LB x 9 hours Purify with Qiagen Miniprep kit, elute in 30 uL water Yielded: ~300 ng x 50 uL of pOpenTaq
- PCR with Stoffel HindIII/EcoRI primers
Hairpin at 61.7C for reverse primer, so try 58C, 60C, and 62C for PCR (previously amplification at 55C was too inefficient) Reaction: 35 uL Phusion Flash 2xMM, 5 uL forward primer, 5 uL reverse primer, 20 uL water, and 5 ul of ~100 ng (total 70 uL) Program: 98C for 10s, [98C for 1s, 55C for 5s, 72C for 30s] x 30 cycles, 72C for 60s, 10C forever
- Agarose gel purify
Made 1% agarose gel 1 lane for 1 kb ladder, 3 lanes for each temperature (58C, 60C, 62C), loaded all of PCR reactions Ran at 120V for 1 hour Perform gel cut for band between 1.5-2kb Perform gel purification with 3x ADB, put into columns once gel have dissolved, eluted in 15 uL water Yielded for 58C: 115.2 ng/uL Yielded for 60C: 148.1 ng/uL Yielded for 62C: 144.6 ng/uL
- Agarose gel of Stoffel PCR
File:Stoffel pcr gel 6-23-14.jpg
- Perform check digestion
Stoffel gene have an NheI digestion site that generates fragments of sizes 1176 and 453 bp. Used 5 uL of PCR products for each temperature Reaction: 5 uL PCR product, 2.5 uL NEB Buffer 2, 0.5 uL NheI, 17 uL water Program: 37C for 40 minutes Ran on 1% agarose gel with 1 kb ladder for 20 minutes Saw three bands: uncut, ~1.1kb, ~500bp.
- Agarose gel of Stoffel digestion
File:Stoffel digest NheI 6-23-14.jpg
- Perform HindIII and EcoRI digest on pUC19 vector and Stoffel insert
Reaction EcoRI: 10.0 uL vector or stoffel insert (use at least 1 ug of the vector) 3.0 uL EcoRI buffer 1.0 uL EcoRI 16.0 uL water Leave at 37C for 1 hour Reaction HindIII: 30.0 uL EcoRI reactionStoffel 7.0 uL HindIII buffer 1.0 uL HindIII 62.0 uL water Leave at 37C for 1 hour Yielded: 25 uL x 30.0 ng/uL of digested Stoffel insert (Qiaquick column purification)
- Agarose gel cut
Made 1% agarose gel Load digestion products Run at 120 V for 60 minutes Cut pUC19 vector at 2.7 kbp Yielded: 15 uL x 79.2 ng/uL of digested pUC19 vector
- Agarose gel of pUC19 vector digest from Colony#17 (wrong insert verified by Sanger sequencing)
- The size of the insert seems to be the same as my Stoffel insert but Sanger sequencing can not give the correct sequence result for this plasmid.
File:PUC19 digest EcoRI HindIII 6-23-14.jpg
- Ligation
Reaction: 100 ng of pUC19 vector ( 1.3 uL x 79.2 ng/uL ) 188 ng of insert ( 6 uL x 30.0 ng/uL ) 1x Ligase buffer ( 5.0 uL x 10x buffer) 600 units T4 DNA Ligase ( 1.5 uL x 400 unit/uL) 36.2 uL of water Program: Room temperature for at least 10 minutes (cohesive sticky ends)
- Transformation into BL21(DE3)
Used all 50 uL ligation reaction to transform 1 tube of competent cells. Incubate on ice for 30 minutes Heatshock 1 minute at 42C Add 250 uL SOC media Incubate on shaker at 37C for 1.5 hr. Spread all of product onto plate Incubate at 37C overnight Yielded: 2 positive colonies.
- Streak 2 colonies onto LB/Amp plate for long-term (6mo) keeping
- Purify colony #1 & #2 plasmids
Make 5 mL overnight liquid cultures.
- Screen colonies using NheI digestion
Screening failed, two colonies were self-ligated pUC19.
September 10, 2014 update[edit]
- Antarctic Phosphatase to prevent self-circularization of vector
(Found some Antarctic Phosphatase in the freezer) Reaction: 100 ng of pUC19 vector ( 1.3 uL x 79.2 ng/uL ) 1x Antarctic Phosphatase Reaction buffer ( 1.0 uL x 10x bufer) 5 units of Antarctic Phosphatase ( 1.0 uL x 5 units/uL) 6.7 uL of water Program: 15 minutes at 37C 5 minutes at 70C to heat inactivate
- Ligation
Reaction: 100 ng of pUC19 vector ( 10 uL of phosphatase reaction ) 188 ng of insert ( 6.0 uL x 30.0 ng/uL ) 1x Ligase buffer ( 5.0 uL x 10x buffer) 600 units T4 DNA Ligase ( 1.5 uL x 400 unit/uL) 27.5 uL of water Program: Room temperature for at least 10 minutes (cohesive sticky ends)
- Transformation into Top10 E. coli
Used all 50 uL ligation reaction to transform 1 tube of competent cells. Incubate on ice for 30 minutes Heatshock 1 minute at 42C Add 250 uL SOC media Incubate on shaker at 37C for 1.5 hr. Spread all of product onto plate Incubate at 37C overnight
- Purify Stoffel & Taq