User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/12

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Contents

Agarose Gel Extraction

Gels were prepared using 0.6g of Agarose powder with 60ml of water, heated until no specks remained and then cooled. 5µl of Ethidium Bromide was added to the gel.


Gel: to extract a doubled digested vector based on the digestion User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/11#Double digestion pUA66 XhoI / BamHI


  1. Lane 1: 10µl 1kb Quick NEB Ladder
  2. Lane 2: 25µl Digest pUA66#A, XhoI/BamHI
  3. Lane 3: 25µl Digest pUA66#A, XhoI/BamHI
  4. Lane 4: Blank
  5. Lane 5: 5µl Digest pUA66#B, XhoI/BamHI
  6. Lane 6: 5µl Digest pUA66#B, XhoI/BamHI
  7. Lane 7: Blank
  8. Lane 8: Blank

Gel Result: Image:12082010-Digestion2x-cut.jpg

Gel Purification

The gel bands were cut and weighted with the following results:

  1. pUA66#A1: 130mg
  2. pUA66#A2: 117mg
  3. pUA66#B1: 162mg
  4. pUA66#B2: 157mg

From this the following measurements where used:

  1. Add 1:3 volume of QG Buffer
    1. pUA66#A1, 130x3 = 390µl QG Buffer
    2. pUA66#A2, 117x3 = 351µl QG Buffer
    3. pUA66#B1, 162x3 = 486µl QG Buffer
    4. pUA66#B2, 157x3 = 471µl QG Buffer
  2. Mix well, place in 47ºC water bath for 10 minutes, mixing ever 2-3 minute to dissolve gel
  3. Add 1:1 volume of Isopropanol
    1. pUA66#A1, 130µl Isopropanol
    2. pUA66#A2, 117µl Isopropanol
    3. pUA66#B1, 162µl Isopropanol
    4. pUA66#B2, 157µl Isopropanol
  4. For each tube place apply to separate column
  5. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  6. For each column add 500µl QG Buffer
  7. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  8. Add 750µl of PE wash with Ethanol
  9. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  10. Centrifuge for an additional minute at 13,000 rpm and discard flow-through
  11. Place each column in a separate Eppendorf tube
  12. For each column add 30µl water and wait for 1 minute
  13. To elute centrifuge for 1 minute at 13,000 rpm, label and keep tube stored at -20ºC until use.


Gel Purification Check

Quality assurance before use in transformation.

  1. Lane 1: 5µl 1kb Quick NEB Ladder
  2. Lane 2: 2µl Digest pUA66#A1, XhoI/BamHI/Purified
  3. Lane 3: 2µl Digest pUA66#A2, XhoI/BamHI/Purified
  4. Lane 4: Blank
  5. Lane 5: 2µl Digest pUA66#B1, XhoI/BamHI/Purified
  6. Lane 6: 2µl Digest pUA66#B2, XhoI/BamHI/Purified
  7. Lane 7: Blank
  8. Lane 8: Blank

Gel Result: Image:12082010-Digestion2x-purified.jpg

Nanodrop

Sample ng/µl 260/280
pUA66#1-11082010-X-B-Purified 5.31 2.01
pUA66#2-11082010-X-B-Purified 6.46 1.86
pUA66#3-11082010-X-B-Purified 15.01 0.98
pUA66#4-11082010-X-B-Purified 8.92 1.81
pUA66#5-11082010-X-B-Purified 12.52 2.11
pUA66#6-11082010-X-B-Purified 9.13 1.92
pUA66#A1-12082010-X-B-Purified 22.63 1.69
pUA66#A2-12082010-X-B-Purified 19.44 1.62
pUA66#B1-12082010-X-B-Purified 8.01 1.80
pUA66#B2-12082010-X-B-Purified 22.55 1.89

Ligation

4 Ligations were setup

  1. 1µl 10xLigation Buffer NEB
  2. 1µl T4 Ligation NEB
  3. 1µl of Template DNA
    1. pUA66#A1, 1µl
    2. pUA66#A1, 1µl
    3. pUA66#B2, 1µl
    4. pUA66#B2, 1µl
  4. For each tube add 8µl katE promoter
  5. Incubate at room temperature for 2 hours

Transformation

Six transformation were setup

  1. Transfer 6 tubes of competent cell straight onto ice for 5 minutes
  2. For each tube add DNA template
    1. pUA66#A1 10 µl
    2. pUA66#A2 10 µl
    3. pUA66#B1 10 µl
    4. pUA66#B2 10 µl
    5. pUA66#A1 (8µl water, 2µl pUA66#A1), without T4
    6. pUA66#4 Undigested, 1µl
  3. Wait for an additional 5 minutes
  4. Place in 42ºC water bath for 1 minute
  5. Place back on ice for 5 minutes
  6. Add 250µl SOC (should be 37ºC)
  7. Incubate in shaker for 1 hour
  8. Add the entire content of each onto a Kanamycin LB-Agar plate and use glass beads to spread
  9. Incubate overnight

PCR, katE

Generate insert using colony PCR on 4 tubes each of 50µl volume

Colony Dillution

  1. Add 10µl water to an Eppendorf tube
  2. Add one colony E.coli
  3. Mix well

Master mix (4x)

  1. 100µl Water
  2. 20µl 10xOptimized DyNAzyme Ext Buffer
  3. 4µl 10mM dNTP
  4. 8µl Primer: HB katE F
  5. 8µl Primer: HB katE R

Reaction

  1. Aliquot 35µl Master Mix into each of four 0.5ml PCR tube
  2. Add 1µl Template DNA from the Colony Dilution
  3. For each tube add 5µl of DyNAzyme EXT DNA Polymerase (possible error - as I might not have added this)
  4. Place in PCR thermocycler

Reaction Environment

Lid: 100ºC; Volume: 40µl

  1. 94ºC for 05:00
  2. 94ºC for 00:30
  3. 55ºC for 00:50
  4. 72ºC for 10:00
  5. GOTO step 2, 32 times
  6. 72ºC for 10:00
  7. 8ºC forever
  8. END

Overnight growth

Inoculate 4 bottles of 10ml LB broth containing 10µl Kanamycin with colonies pUA66

  1. Add 10µl Kanamycin 50mg/ml to each bottle of LB-broth
  2. Swab and add 1 colony of pUA66 from plate
  3. Place in shaker overnight at 37ºC

Transformation of Ligation

Perform at total of 6 transformation which includes self-ligation and viability test.

Before: Place 2 bottles of 1ml SOC in a 37ºC incubator

  1. Collect 6 bottles of competent cell from the -80ºC freezer and place on ice for 5 minutes
  2. Add the plasmid / ligation product
    1. Tube A1, add the complete ligation reaction pUA66katE#A1
    2. Tube A2, add the complete ligation reaction pUA66katE#A2
    3. Tube B1, add the complete ligation reaction pUA66katE#B1
    4. Tube B2, add the complete ligation reaction pUA66katE#B2
    5. Tube S (Self-ligation test), add 2µl of digested plasmid pUA66#A1
    6. Viability Test, add 1µl of pUA66 undigested plasmid
  3. Wait for 5 minutes to allow mixture to settle
  4. Place each tube in a 42ºC water bath for 1 minutes
  5. Place the cells back on ice and wait 5 minutes
  6. For each tube add 250µl SOC (warm 37ºC)
  7. Place the tubes in a shaker at 37ºC for 1 hour
  8. Pour the complete mixture onto plates containing Kanamycin
  9. Spread using glassbeads
  10. Incubate overnight at 37ºC
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