User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/20

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Overnight growth check

Great! All four bottles grew well and remained intact.

Glycerol stock

After the previous day mistake - the first thing to be done was to take a glycerol stock from each tube and label accordingly.

For each bottle I prepared stock as follows:

  1. 700µl broth with growth
    1. pUA66katE#A1#1
    2. pUA66katE#A1#2
    3. pUA66katE#A1#3
    4. pUA66katE#A1#4
  2. 300µl 50% Glycerol
  3. Store at -80ºC

Miniprep

After taking the stock I then moved onto centrifuging the bottles to extract the plasmids

  1. Spin all bottles for 10 minutes in centrifuge
  2. Discard supernatant
  3. Place the bottles in an icebucket for miniprep

For each of the bottles:

  1. Resuspend in 500µl cold P1 buffer
  2. Distribute 250µl in two eppendorf tubes
  3. For each tube add 250µl P2 Lysis buffer and wait 4 minutes
  4. Add 350µl N3 neutralisation buffer
  5. Centrifuge at 13,000 rpm for 10 minutes
  6. Apply the first tube to a QIAgen Quick column
  7. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  8. Apply the second tube to the column
  9. Centrifuge for 1 minute at 13,000 rpm and discard flow-through, all the plasmids is now bound to the column
  10. For each column add 500µl PB wash buffer
  11. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  12. For each column add 750µl PE with Ethanol final wash buffer
  13. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  14. Centrifuge for an additional minute at 13,000 rpm and discard flow-through
  15. Place columns in each labelled Eppendorf tubes
  16. Add 30µl of water to the centre of the column and wait for 1 minutes
  17. Centrifuge for 1 minute at 13,000 rpm to elute plasmid
  18. Store plasmids in an icebucket (or in the freezer at-20ºC)

Digestion

With the plasmids ready, I moved straight onto a double digestion.

Master mix: (total volume of 80µl)

  1. 2µl 100xBSA Buffer
  2. 20µl 10xNEB#Buffer
  3. 4µl XhoI
  4. 4µl BamHI
  5. 50µl water

Reaction: (total volume of 50µl each)

  1. Aliquot 20µl into each tube of 30µl DNA
  2. Incubate for 2 hours at 37ºC


Gel Purification p67

The products were then purified using a QIAgen Gel Extraction Kit and eluted in 30µl water.

  1. Add 1:3 Volume QG Buffer
    1. p67, 480µl
  2. Place in 47ºC water bath for 10 minute, mix ever 2 minute to dissolve gel
  3. Add 1:1 Volume Isopropanol
    1. p67, 160µl
  4. Apply each tube to a separate column
  5. Centrifuge for 1 minute at 13,000 rpm, discard supernatant
  6. Apply remainder ( it was getting a bit full) for each tube to each related column
  7. Centrifuge for 1 minute at 13,000 rpm, discard supernatant
  8. Add 500µl QG Buffer
  9. Centrifuge for 1 minute at 13,000 rpm, discard supernatant
  10. Add 750µl PE Final wash with Ethanol
  11. Centrifuge for 1 minute at 13,000 rpm, discard supernatant
  12. Centrifuge for an additional minute at 13,000 rpm, discard supernatant
  13. Place columns in labelled eppendorf tubes
  14. Apply 30µl water to the center of each columns and wait for 1 minute
  15. To elute, centrifuge for 1 minute at 13,000 rpm and keep tube

2% Gel of Digestion

I could now rescreen all the colonies from pUA66katE#A1 digestions on a 2% gel

Gel 2%

  1. Add 1.2g Agarose powder
  2. Add 60ml 1XTAE
  3. Heat in microwave until no specs and cool
  4. Add 2µl Ethidium Bromide
  5. Pour in prepared tray

Loading:

  1. Lane 1: 10µl, 100bp NEB Ladder
  2. Lane 2: 2µl, p67, XhoI/BamHI, purified
  3. Lane 3: Blank
  4. Lane 4: 50µl, pUA66katE#A1#4, XhoI/BamHI
  5. Lane 5: 50µl, pUA66katE#A1#3, XhoI/BamHI
  6. Lane 6: 50µl, pUA66katE#A1#2, XhoI/BamHI
  7. Lane 7: 50µl, pUA66katE#A1#1, XhoI/BamHI
  8. Lane 8: Blank


Gel Picture:

Analysis: Lane 6, containing sample pUA66katE#A1#2 contains the insert!!!

Transformations from overnight ligation

This was a transformation using the overnight ligation (4 SAP and 2 standard)

Before:

  1. Preheat 1ml SOC in incubator 37ºC
  2. Place 6 Kanamycin plates in incubator 37ºC to dry
  3. Label plates

Procedure:

  1. Collect 6 tubes of 50µl XL1-Blue competent cells from -80ºC and place on ice
  2. Keep on ice for 5 minutes
  3. For each tube of cells, label and add one of the reaction (std ligation and sap ligation)
  4. Keep on ice for another 5 minutes
  5. Place in a 42ºC water bath for 1 minute
  6. Place back on ice for 5 minutes
  7. Add 250µl of SOC for each bottle
  8. Incubate in shaker at 37ºC for 15 minutes
  9. For each bottle apply the liquid to the centre of the plate and spread using glassbeads
  10. Incubate overnight at 37ºC

Ligation p67

I setup a quick reactions in a standard ligation 1:7

  1. 1µl Ligation Buffer
  2. 1µl T4
  3. 1µl p67 XhoI/BamHI
  4. 7µl katE XhoI/BamHI
  5. Incubated for 1 hour at 37ºC

Transformation p67

This was a transformation using the quick ligation of p67

Before:

  1. Preheat 1ml SOC in incubator 37ºC
  2. Place 1 Kanamycin plate in incubator 37ºC to dry
  3. Label plate

Procedure:

  1. Collect 1 tube of 50µl XL1-Blue competent cells from -80ºC and place on ice
  2. Keep on ice for 5 minutes
  3. Add all of the p67 ligation reaction
  4. Keep on ice for another 5 minutes
  5. Place in a 42ºC water bath for 1 minute
  6. Place back on ice for 5 minutes
  7. Add 250µl of SOC for each bottle
  8. Incubate in shaker at 37ºC for 15 minutes
  9. For each bottle apply the liquid to the centre of the plate and spread using glassbeads
  10. Incubate overnight at 37ºC


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