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