User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18

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Contents

Check overnight growth 4 bottles of pUA66katE and 2 bottles of pBestLuc

These cultures are from the previous days ligation and plasmid transformation plates grown overnight in 10ml LB (pUA66katE, on Kanamycin and pBestLuc on Ampicillin).

All bottles grew well:)

Prepare stock of pBestLuc

For one of the pBestLuc bottles I prepared a stock solution.

  1. Add 700µl of overnight growth to an Eppendorf tube
  2. Add 300µl 50% Glycerol
  3. Store at -20ºC

Note: I need to move these stocks into -80ºC (Done, 19 August 2010)

Miniprep 2 bottles pUA66katE and 1 bottle pBestLuc

I used 3 bottles for miniprep:

  1. Spin all bottles for 10 minutes in centrifuge
  2. Discard supernatant
  3. Store 3 of the bottles at -20ºC (Two of the pUA66katE and one pBestLuc)
  4. Place the remaining 3 bottles in an icebucket for miniprep

For each of the 3 remaining 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)


Setup PCR katE (2 reactions)

To ensure no contamination occurred previously. I setup a new PCR batch with using a fresh set of water.

Colony PCR on 2 tubes each of 41µl volume

Colony Dilution

  1. Add 10µl water to an Eppendorf tube
  2. Add one colony E.coli <- USING COLONIES FROM DIFFERENT BATCH THAN pUA66 (using pBestLuc)
  3. Mix well

Master mix (2x)

For a final volume of 140µl (2x35µl):

  1. 50µl Water
  2. 10µl 10xOptimized DyNAzyme Ext Buffer
  3. 2µl 10mM dNTP
  4. 4µl Primer: HB katE F
  5. 4µl Primer: HB katE R

Reaction

  1. Aliquot 35µl Master Mix into each of two 0.5ml PCR tube
  2. Add 1µl Template DNA from the Colony Dilution
  3. For each tube add 5µl of DyNAzyme EXT DNA Polymerase
  4. Place in PCR thermocycler

Reaction Environment

Lid: 100ºC; Volume: 41µl

  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

Check plate Ligation pUA66katE & new plasmid

All plates had grown overnight. The ligation again showed small transparent colonies for the pUA66katE.

The new plasmid looked promising and will be used to check the ligation, e.g. if it is the vector I am using that is prone to self-ligation.

I am not sure if there is some sort of star activity causing none-specific binding.

Setup 2xDigestion pUA66katE B-X and one in a series (first X and then B, to run overnight)

Two digestions were set up.

Double digestion, total volume of 50µl

  1. 30µl pUA66katE#1
  2. 0.5µl, 100xBSA
  3. 5µl, 10xNEB#3
  4. 1µl XhoI
  5. 1µl BamHI
  6. 12.5µl Water
  7. Incubate for 2 hours at 37ºC.

Single digestion, total volume of 50µl

  1. 30µl pUA66katE#1
  2. 0.5µl, 100xBSA
  3. 5µl, 10xNEB#3
  4. 1µl XhoI
  5. 13.5µl water
  6. Incubate for 5 hours at 37ºC.


Quick digestion of PCR

With the PCR ready, I will now try to do the digest directly on the one reaction. I want to try a real short digest.

For a final volume of 60µl

  1. 50µl katE raw PCR
  2. 0.6µl, 100xBSA
  3. 6µl, 10xNEB#3
  4. 1µl XhoI
  5. 1µl BamHI
  6. 3.4µl water
  7. Incubate for 15 minutes at 37ºC.

Gel, 2% PCR(katE) and insert screening (pUA66katE)

In this step I will run the ready 2xDigested pUA66katE and my PCR products 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: 50µl, pUA66katE#1, XhoI/BamHI
  3. Lane 3: Blank
  4. Lane 4: 60µl, katE, XhoI/BamHI
  5. Lane 5: Blank
  6. Lane 6: 50µl, katE, undigested
  7. Lane 7: Blank
  8. Lane 8: 5µl, 1k NEB Quick Ladder (pointless)

I cut the gel before taking the gel picture.

Gel Picture: Image:18082010-2percent-plasmid-insert.jpg

Analysis: Lane 2 shows aspects of genomic contamination - however there is also a distinct band at 400bp. This could most certainly be my insert!!!

Cutting from 2% Gel

As I needed the PCR products and had thought the digested pUA66kate#1 failed, I decided to also cut the vector band.

Using a scalpel, I cut the following:

Sample Weight (mg) Size (bp)
pUA66kate#1 XhoI/BamHI 150 4260
katE XhoI/BamHI 170 400
katE Undigested 210 400

Gel purfication, pUA66katE XhoI/BamHI and katE XhoI/BamHI

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

  1. Add 1:3 Volume QG Buffer
    1. katE2, 660µl
    2. katE3, 630µl
    3. katE4, 729µl
  2. Place in 47ºC water bath for 10 minute, mix ever 2 minute to dissolve gel
  3. Add 1:1 Volume Isopropanol
    1. katE2, 220µl
    2. katE3, 210µl
    3. katE4, 243µl
  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

Check purified products on a 1% Gel

In order to be able to use the digests for ligation I ran the samples on a 1% Gel

Gel 1%

  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, 1kb NEB Quick Ladder
  2. Lane 2: 5µl, pUA66katE#1, XhoI/BamHI, purified
  3. Lane 3: 5µl, katE, XhoI/BamHI, purified
  4. Lane 4: 5µl, katE, undigested, purified
  5. Lane 5-8: Blank

Gel Picture: Image:18082010-2percent-plasmid-and-inserts.jpg

Analysis: Lane 2, is now cleaned up from the purification but is coming up with two bands! One possibility is that the top is 4660bp (4260 vector+400 insert) whilst the one below is 4260 (vector only).

Prepare 8 plates kanamycin

I prepared 8 plates with kanamycin by

  1. Heat 200ml of autoclaved LB Agar
  2. Add 200µl of 50mg/µl Kanamycin stock
  3. Pour each plate with around 20ml
  4. Wait for 1 hour

Overnight Growth (pUA66katE and plasmid 67)

I prepared 6 bottles of 10ml LB Broth and add 10µl of 50mg/µl Kanamycin stock.

  1. 4 Bottles were setup based on picking colonies from this morning ligated plate pUA66katE
  2. 2 bottles where setup based on picking two colonies from the new plasmid 67
  3. The bottles were placed in a shaker at 37ºC overnight

Do a miniprep of the 2 remaining bottles

Seeing that there was a potential result from Lane 2 and without having the glycerol stock. I proceeded to miniprep the overnight growth (pellets) from the remaining 2 colonies and do a plasmid transformation using 1µl from each, keeping 5µl of plasmid in case and then doing a double digestion overnight using the remaining 24µl.

The miniprep followed exactly the same protocol as earlier, but I had a labelling issue which may have mixed the second part of the two samples.

  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 <=== Potential mix
  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)


Overnight Digestion (pUA66katE#A1#3 and pUA66katE#A1#4)

I setup two double digests overnight from the new minipreps using 24µl DNA, 5µl was set aside and stored at -20ºC.

The digests were setup as follows.

Digest #3

  1. 24µl pUA66katE#A1#3
  2. 0.5µl 100xBSA
  3. 5µl 10xNEB Buffer#3
  4. 1µl XhoI
  5. 1µl BamHI
  6. 19.5µl water
  7. Incubate at 37ºC Overnight

Digest #4

  1. 24µl pUA66katE#A1#4
  2. 0.5µl 100xBSA
  3. 5µl 10xNEB Buffer#3
  4. 1µl XhoI
  5. 1µl BamHI
  6. 19.5µl water
  7. Incubate at 37ºC Overnight

Grow pUA66katE#A1

I put the plate pUA66katE#A1 back in the incubator to see if the colonies would regrow and to re-screen for the colony that had come up with the insert.

Plasmid Transformation (pUA66katE#A1#3 and pUA66katE#A1#4)

This was a 15 minute quick transformation using 1µl of plasmid from the Miniprep (#3 and #4)

Before:

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

Procedure:

  1. Collect 2 bottles of 50µl XL1-Blue competent cells from -80ºC and place on ice
  2. Keep on ice for 5 minutes
  3. Add 1µl of plasmid for each of the bottles (bottle#3 and bottle#4)
  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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