From 2007.igem.org

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Addition of prefix and suffix

A large ammount of time was spent getting this ligation to work. We believe this was due to problems with the digestion of the PstI site both at the end of the suffix and in BJa61035 where it becomes close to the end after cleavage of the XbaI site. A number of factors contribute to this:

• The site is surrounded by CG pairs due to our use of a GC clamp, and NotI sites.
• The site is close to the end of the DNA.
• The ligation is ineficient due to the karge plasmid size.

What follows is a description of our best practice, that finally yielded a result. The gory details of the 53 experimental conditions we tried to get to this and the gels are available here.

sequences for prefix and suffix primers

• Diluted DNA from last round to give 10ng/ul

Tube	conc of miniprep ng/ul total=(100ul)	x uL of miniprep added to y mL milliQ
41C	129	1ul in 10.9ul

set up PCR using quickchangeXL kit components:

• 50uL reaction containing:
  • 5uL of x10 buffer
  • 2uL of 10ng/uL template (41C)
  • 2uL of GvpB-forward primer (BF) (at 125ng/2uL)
  • 2uL of GvpU-reverse primer (UR) (at 125ng/2uL)
  • 1uL of dNTP
  • 3uL of quickchange
  • 35ul of milliQ water
  • 1uL of Pfu Ultra.

• PCR Cycle:
  • segment 1 : 95degC for 2 minutes x1
  • segment 2 : x3
    • 95 degC for 2 minute
    • 55 degC for 1 seconds
    • 68 degC for 15 minutes
  • segment 3 : x15
    • 95 degC for 2 minute
    • 60 degC for 50 seconds
    • 68 degC for 15 minutes
  • segemnt 5 : 68 degC for 15 minutes
  • segment 5 : 4 degC indefinitely

• Prepare insert:
  • Digest pcr product with 1uL of DpnI (quickchange XL kit) 1hr at 37 deg C.
  • Gel purify whole reaction using promega kit giving 40uL in TE.
  • Digest purified pcr/dpnI product with 2uL of XbaI, 2uL of SpeI 6uL bSA X10 and 6uL Buffer2 x10 for 2hours at 37 deg C
  • Gel purify with promega kit. (only one band present)
  • gives about 40uL @ 8ng/ul in TE
• Preppare vector
  • Digest 5uL(252ng/ul) of BBa_J61035 with 1uL of SpeI, 2uL Buffer2 x10, 2uL BSA x10 for 2 hours at 37 deg C.
  • Treat BBa_J61035 digest with antarctic phosphatase 1uL and 2uL of it's buffer. 70minutes at 37 deg.
  • Heat inactivate 80degC for 20minutes.
  • Gel purify with promega kit (only one band present)
  • gives about 40u @ 63ng/ul in TE
  • Dilute 1 in 5 to give working solution at 12ng/uL
• Ligation:(11.5uL reaction)
  • 5 ul of 5 minute ligation buffer x2
  • 4.0 uL of insert
  • 1.5uL of vector
  • 1.0 uL of T4 ligase. added when all at room temperature.
  • leave at room temperature for five minutes then return to ice.
• Transformation
  • Follow Igem transformation protocol for 50uL of DH5a cells, using 8uL of the ligation mixture.
• Plate onto LB amp plates.
• Pick colonies for overnight culture.
• Minipreped remaining culture (4mL) giving 100uL.
• Digest with EcoRI 1uL,SpeI 1uL, PstI 1uL in buffer 2 and BSA
• Screen: Note these colonies can load the insert in one of two orientations.Depending on the orientation the SpeI restriction site will be reconstituted at one or other end of the insert. The correct orientation can be determined because if the S site reconstitutes correctly at the end then this diggest will increase leave the 1500bp gentamycin cassette attached to the part. If the part goes in the other way the Spe site is reconstructed between the cassette and part and both band s appear.

Results: No insert control gave a plate with 4 colonies. (0 in 4 sqcm, value in excell spead sheet) Ligation plate gave a plate with 25 colonies. (5 in 4sqcm, value in excel spread sheet)