User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13

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Plates

Plate result from transformation User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/12#Transformation of Ligation


Plate A1: pUA66katE Ligation; 12/08/2010; 50-70 colonies

Plate A2: pUA66katE Ligation; 12/08/2010; 50-70 colonies


Plate B1: pUA66katE Ligation; 12/08/2010; 50-70 colonies

Plate B2: pUA66katE Ligation; 12/08/2010; 50-70 colonies

Plate STD: pUA66 undigested; 12/08/2010; (Viability Test); 1000 colonies

Plate S: pUA66 contains no T4 (Self-Ligation Test); 12/08/2010; 0 colonies

Digestion of katE insert

Double digestion using BamHI / XhoI on PCR product User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/12#PCR, katE

Master mix

For 4 tubes of 40µl PCR product the master mix was setup as follow:

  1. 20µl 10xNEB#3
  2. 2µl 100xBSA
  3. 4µl XhoI
  4. 4µl BamHI

Reaction

  1. Aliquot 10µl into each of the 4 PCR product tubes already containing 40µl
  2. Place in incubator for 2 hours

Gel of digested PCR product

Prepare a 2% Gel run and cut the product

2% Gel

  1. Add 1.2g Agarose Powder
  2. Add 60ml of 1xTAE Buffer
  3. Heat using a microwave until all specks have cleared
  4. Pour in tray

Loading

  1. Lane 1: 10µl of 100b NEB Ladder
  2. Lane 2: 50µl of PCR tube 1
  3. Lane 3: 50µl of PCR tube 2
  4. Lane 4: 50µl of PCR tube 3
  5. Lane 5: 50µl of PCR tube 4
  6. Lab 6-8: BLANK

Run at 100V, 400mA for 60 minutes


Gel Result

Error - no band on gel! --- STOP / Restarting protocol !!! Arghhh what a shame - a beautiful gel gone to waste

Miniprep

Miniprep of overnight growth User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/12#Overnight growth

The four tubes of 10 ml took some time to grow - but they were put on rather late the night before.

Below uses the QIAgen miniprep kit and is adjusted for low-copy number plasmids

  1. Spin all 10ml tubes for 10 minutes at 3,600rpm
  2. Remove supernatant
  3. Resuspend cells in 500µl P1 buffer
  4. Aliquote 250µl into eppendorf tubes, label pairs (e.g. A-A, B-B..)
  5. For each tube, Add 250µl of P2 - lysis buffer - and wait for 4 minutes for lysis to occur
  6. For each tube, Add 350µl of N3 - neutralisation buffer - and invert immediately 12 times
  7. Centrifuge for 10 minutes at 13,000 rpm
  8. Apply supernatant of the first pair (A-B-C-D) onto columns
  9. Centrifuge for 1 minute at 13,000 rpm discard flow-through
  10. Apply the supernatant of the second pair of tubes to the columns
  11. Centrifuge for 1 minute at 13,000 rpm discard flow-through
  12. To each of the four columns add 500µl PB wash
  13. Centrifuge for 1 minute at 13,000 rpm discard flow-through
  14. To each of the four columns add 750µl PE final wash with Ethanol
  15. Centrifuge for 1 minute at 13,000 rpm discard flow-through
  16. Centrifuge for an additional minute at 13,000 rpm discard flow-through
  17. To elute, apply 30µl of Water to the centre of the column and wait for 1 minute
  18. Centrifuge for 1 minute at 13,000 rpm, label and keep tube on ice (or at -20ºC for long time storage)

Digestion of Miniprep

Double digestion using BamHI / XhoI on pUA66 Miniprep User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Miniprep

Master mix

For 4 tubes of 30µl miniprep plasmids (pUA66) the master mix was setup as follow:

For a final volume of 40µl (4x10µl)

  1. 20µl 10xNEB#3
  2. 2µl 100xBSA
  3. 4µl XhoI
  4. 4µl BamH
  5. 10µl Water

Reaction

  1. Aliquot 10µl into each of the 4 miniprep tubes already containing 30µl plasmid
  2. Place in incubator for 2 hours

Restart PCR, katE

After the earlier PCR flop User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Gel of digested PCR product, I decided to restart a reaction. I am here paying attention to two details that may have caused the error!

  1. Check that colonies are really going into the colony dilution
  2. Ensure that the last step - adding Polymerase is done!

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

Colony Dilution

  1. Add 10µl water to an Eppendorf tube
  2. Add one colony E.coli <- ENSURE THAT SUFFICIENT COLONY IS TRANSFERED
  3. Mix well

Master mix (4x)

For a final volume of 140µl (4x35µl):

  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 <- SECOND STEP CHECK - ENSURE THAT THIS STEP IS DONE
  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

Overnight growth

To complete the insert check of colonies Editing User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Plates I need to do a miniprep and digestion. If it all works well - I should see a 400bp product. With a successful transformation on multiple plates I decided to do a cross check. I am suspicious that some plates contain self-ligated products or mega-vectors resulting from not having a proper digest prior to ligation.

Inoculate 12 bottles of 10ml LB broth containing 10µl Kanamycin as follows:

  1. Add 10µl Kanamycin 50mg/ml to each bottle of LB-broth
  2. Swab and add 1 colony
    1. pUA66katE#A1 to each of three bottles
    2. pUA66katE#A2 to each of three bottles
    3. pUA66katE#B1 to each of three bottles
    4. pUA66katE#B2 to each of three bottles
  3. Place in shaker overnight at 37ºC

Let's hope for the best:)

Setup 2 Gels, 1% and 2% to cut digested miniprep and PCR product

Prepare a 2% Gel to cut the product (undigested) from the earlier PCR Editing User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Restart PCR, katE.

Prepare a 1% gel to cut out the previous digestion of the miniprep Editing User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Digestion of Miniprep.

2% Gel

  1. Add 1.2g Agarose Powder
  2. Add 60ml of 1xTAE Buffer
  3. Heat using a microwave until all specks have cleared
  4. Pour in tray

1% Gel

  1. Add 0.6g Agarose Powder
  2. Add 60ml of 1xTAE Buffer
  3. Heat using a microwave until all specks have cleared
  4. Pour in tray

Loading 2%

  1. Lane 1: 10µl of 100b NEB Ladder
  2. Lane 2: 41µl of PCR tube 1
  3. Lane 3: 41µl of PCR tube 2
  4. Lane 4: 41µl of PCR tube 3
  5. Lane 5: 41µl of PCR tube 4
  6. Lab 6-8: BLANK

Gel Extraction of katE and pUA66

Loading 1%

  1. Lane 1: 10µl of 1kb Quick Ladder
  2. Lane 2: 40µl of digested pUA66 BamHi/XhoI
  3. Lane 3: 40µl of digested pUA66 BamHi/XhoI
  4. Lane 4: 40µl of digested pUA66 BamHi/XhoI
  5. Lane 5: 40µl of digested pUA66 BamHi/XhoI
  6. Lab 6-8: BLANK

Run both gels at 100V, 400mA for 60 minutes

GREAT! Both gels digested completely and all bands showed up. Still it remains that the vector is very weak in concentration.

Gel Result 1%

Gel Picture 1% (taken 16 AUG 2010)

Gel Result 2%

Gel Picture 2% (taken 16 AUG 2010) Cut

I weighed out 8 tubes before and proceeded to cut the gels under UV light using a scalpel. The gels bands resulted in the following weight:

  1. katE#1: 122mg
  2. katE#2: 147mg
  3. katE#3: 142mg
  4. katE#4: 107mg
  5. pUA66-X/B#1: 58mg
  6. pUA66-X/B#1: 222mg
  7. pUA66-X/B#1: 267mg
  8. pUA66-X/B#1: 187mg

ERROR: As I was working in the dark I placed some of the cut bands in the same bottles - I corrected this after and hope it didn't cause any anomalies (e.g. mix katE and pUA66-X/B).

Gel Purification

From this the following measurements where used: (TODO: check and fix calculation)

  1. Add 1:3 volume of QG Buffer
    1. katE#1: 122x3 = 390µl QG Buffer
  2. #katE#2: 147x3 = 390µl QG Buffer
    1. katE#3: 142x3 = 390µl QG Buffer
    2. katE#4: 107x3 = 390µl QG Buffer
    3. pUA66-X/B#1: 58x3 = 390µl QG Buffer
    4. pUA66-X/B#1: 222x3 = 390µl QG Buffer
    5. pUA66-X/B#1: 267x3 = 390µl QG Buffer
    6. pUA66-X/B#1: 187x3 = 390µl QG Buffer
  3. Mix well, place in 47ºC water bath for 10 minutes, mixing ever 2-3 minute to dissolve gel
  4. Add 1:1 volume of Isopropanol
    1. katE#1: 122µl Isopropanol
  5. #katE#2: 147µl Isopropanol
    1. katE#4: 107µl Isopropanol
    2. pUA66-X/B#1: 58µl Isopropanol
    3. pUA66-X/B#1: 222µl Isopropanol
    4. pUA66-X/B#1: 267µl Isopropanol
    5. pUA66-X/B#1: 187µl Isopropanol
  6. For each tube of katE apply to separate column, however for the 4 tubes of pUA66-X/B use only two columns
  7. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  8. For the pUA66-X/B add the remaining two tubes to each column, centrifuge only these for 1 minute at 13,000 rpm and discard flow-through
  9. For each column add 500µl QG Buffer
  10. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  11. Add 750µl of PE wash with Ethanol
  12. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  13. Centrifuge for an additional minute at 13,000 rpm and discard flow-through
  14. Place each column in a separate Eppendorf tube
  15. For each column add 30µl water and wait for 1 minute
  16. To elute centrifuge for 1 minute at 13,000 rpm, label and keep tube stored at -20ºC until use.

Digestion of Purified katE product

Double digestion using BamHI / XhoI on the purified PCR product User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Gel Purification

Master mix

A master mix was setup to accommodate for 4x30µl of PCR products as follows:

For a final volume of 40µl (4x10µl)

  1. 20µl 10xNEB#3
  2. 2µl 100xBSA
  3. 4µl XhoI
  4. 4µl BamH
  5. 10µl Water

Reaction

  1. Aliquot 10µl into each of the 4 purified PCR product tubes already containing 30µl plasmid
  2. Place in incubator overnight


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