User:DrJones1935/14 August 2012

From 2012.igem.org

Contents

I. Check Plates for Growth

RESULTS:

Growth on positive controls was a large number of colonies but not a lawn as expected. No growth on any of the Tetracycline plates.

II. Transform WT ADP1 using natural competence

  • Inoculate 20 μL of the O/N cultures from yesterday into 300 μL of LB for 4 tubes as follows:
Number Name
1 ADP1 (-)
2 ADP1 +pBAV1K
3 ADP1 +1:1 ligation
4 ADP1 +1:3 ligation
  • Incubate with shaking on its side (using tape to keep the tube in place) at 30 oC for 2.5 hours
  • Add 2.5 μL of ~200 ng pBAV1K/μL sample for ADP1+pBAV1K, and 2.5 μL of sterile H2O for ADP1 (-)
  • Add whatever was left from the ligation mixtures to the two ligation tubes (4-8 uL)
  • Incubate with shaking as above for 2 hours
  • For each sample, add 150 uL of cells to appropriate plate as follows using glass beads
Sample Plate
Negative Control 1 (-) LB + Kan50
Negative Control 2 (-) LB + Tet20
pBAV1K LB + Kan50
pBAV1K Positive Control LB
1:1 Ligation LB + Tet20
1:1 Ligation Positive Control LB
1:3 Ligation LB + Tet20
1:3 Ligation Positive Control LB
  • Incubate at 34 oC until colonies form

III. Digest pBAV1K-T5-gfp Plasmid and Cassette with XbaI and PstI, and CIP for pBAV1K

NOTE: The plasmid digestion was started an hour before the cassette digestion so that the two digestions would end at the same time.

  • Mix (plasmid):
  • 30.5 uL water
  • 10 uL pBAV1K-T5-gfp DNA (~2 ug at 200 ng/uL)
  • 0.5 uL 100x BSA
  • 5 uL 10 NEB Buffer 3
  • 2 uL XbaI
  • 2 uL PstI
  • Digest for 4 hours at 37 oC, then add 0.5 uL of CIP
  • Incubate for another hour at 37 oC
  • Mix (cassette):
  • 40.5 uL cassette DNA (~1.8 ug at 45 ng/uL)
  • 0.5 uL 100x BSA
  • 5 uL 10 NEB Buffer 3
  • 2 uL XbaI
  • 2 uL PstI
  • Digest for 4 hours at 37 oC

IV. PCR for More Cassette

  • Mix Reagents:
  • Mix according to the following table
Negative Control Reactions (x4)
Reagent (uL) (uL)
water 17.25 16.25
5x Fidelity Buffer 5 5
10 mM dNTP mix 0.75 0.75
10 uM primer (F) 0.75 (pBf) 0.75 (pBf)
10 uM primer (R) 0.75 (p2Br) 0.75 (pBr)
Template ("Cassette 1") - 1
KAPA HiFi polymerase 0.5 0.5
  • Run PCR
Step Temp (oC) Time
1 94 5 m
2 98 20 s
3 63 15 s
4 72 5 m
5 GOTO 2 34x
6 72 5 m
7 4

V. AGE of Digestion and PCR Samples

  • Make Gel:
  • Measure out 0.40660 g of agarose and add to a 250 mL E-flask
  • Add 50 mL of 0.5x TBE buffer and swirl to mix (0.8% gel)
  • Cover flask with a Kimwipe and microwave for ~1 minute until clear
  • Allow to cool to ~60 oC and pour into beaker for ethidium bromide (EtBr)
  • Add 1 uL of EtBr stock to agarose and swirl to mix
  • Immediately pour gel into tray with combs
  • Allow to solidify, then remove the comb and tape and place the tray in the gel box with the wells closer to the black electrode
  • Make sure it is submerged in TBE buffer
  • Load Gel:
  • Mix samples as drops on a piece of parafilm
  • Mix:
NEB 2-log (Invtrogen 1 kb plus) Sample
*4 uL pre-mixed *6.3 uL dH2O
*1.7 uL 6x Loading Dye
*2.0 uL DNA
  • Load ~10 uL on gel according to the following chart (except ladder which is 4 uL):
Lane 1 2 3 4 5 6 7 8
2-log ladder Plasmid Digestion Cassette Digestion (-) C1 C2 C3 C4
  • Store DNA at 4 oC
  • Unusual well formation in some cases
  • Run Gel:
  • Close gel box and turn on power pack
  • Run gel at 30 V for ~50 min to stack gel
  • Run gel at 75 V until the markers have reached ~3/4 of gel
  • Image Gel:
  • RESULTS:
Srk 2012-08-14 19hr 34min.jpg

Source: Media:Srk_2012-08-14_19hr_34min.tif

Feature Expected Size (bp)
Uncut Cassette 5152
Cut Cassette 5144
Uncut Plasmid 3653 (circular)
Cut Plasmid 2807
GFP fragment 846

VI. Prepare for Gel Extraction of Cassette

  • Make Gel:
  • Measure out 0.40460 g of agarose and add to a 250 mL E-flask
  • Add 50 mL of 0.5x TBE buffer and swirl to mix (0.8% gel)
  • Cover flask with a Kimwipe and microwave for ~1 minute until clear
  • Allow to cool to ~60 oC
  • Immediately pour gel into tray with comb
  • Allow to solidify, then remove the comb and tape and place the tray in the gel box with the wells closer to the black electrode
  • Make sure it is submerged in fresh TBE buffer
  • Load Gel:
  • Add 9.6 uL of Loading Dye to each of the ~48 uL samples and mix.
  • Load on gel according to the following chart:
Lane 1 2 3 4 5 6 7
NEB 2-log ladder (4 uL, premixed) Plasmid Digestion (~9.6 uL) Plasmid Digestion (~48 ul) empty NEB 2-log Cassette Digestion (~9.6 uL) Cassette Digestion (~48 ul)
  • Run Gel:
  • Close gel box and turn on power pack
  • Run gel at 30 V for 20 min
  • Run gel at 75 V until the markers have reached 3/4 of gel
  • Cut Gel:
  • Remove gel from box and cut along lanes to separate the 9.6 uL lanes from the 48 uL lanes.
  • Return 48 uL lanes to the gel box
  • Post-stain with EtBr
  • Place 9.6 uL lanes in 200 mL of 0.5 ug/mL EtBr solution for 15 minutes
  • Image Gel
  • View 10 uL lane under UV to identify band of interest
  • Use a clean razor blade to nick gel where the band is
  • Excise Gel Fragments
  • Match 48 uL lanes to the 9.6 uL lanes
  • Using the 9.6 uL lane as a guide, cut out the band from the 48 uL lane

VII. QIAquick Gel Extraction Protocol

  • Weigh each gel slice in a colorless 15 mL conical tube:
Plasmid Digestion Cassette Digestion
378 mg 399 mg
  • Add 3 volumes Buffer QG to 1 volume of gel
Plasmid Digestion Cassette Digestion
1134 uL 1197 uL
  • Incubate in 50 oC water bath for 12 minutes until all gel has dissolved. Vortex to help mix.
  • Note: The solution was yellow, OK to proceed
  • Add 1 volume 100% isopropanol to samples and mix by inversion
Plasmid Digestion Cassette Digestion
378 uL 399 uL
  • Apply each sample to a QIAquick column 800 uL at a time. Centrifuge for 1 minute at 13000 rpm, discard flow-through, and repeat until all of the sample has passed through the column
  • Add 500 uL of Buffer QG to the QIAquick columns and centrifuge for 1 minute, discard flow-through
  • Add 750 uL of Buffer PE to the QIAquick column and incubate on the bench for 5 minutes
  • Centrifuge for 1 min and discard flow through
  • Centrifuge again for 1 minute
  • Transfer the columns to clean microcentrifuge tubes
  • Elute DNA by adding 30 uL of sterile water to the center of each column and let stand for 4 minutes
  • Centrifuge for 1 minute