User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/17

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Result overnight growth pUA66katE Ligation

  1. pUA66katE, 4 colonies - see User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/16#Transformation, Ligation pUA66katE
  2. pBestLuc, lawn of colonies -see User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/16#Transformation, pBestLuc

Miniprep

I will miniprep the remaining 2 bottles from the ligased pUA66katE#A1 plate User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Plates

  1. Add 500µl P1 buffer to resuspend by pipetting
  2. For each bottle aliquote 350µl in two labelled eppendorf tubes
  3. For each eppendorf tube (4 in total) add 350µl P2 Lysis buffer and wait for 4 minutes
  4. For each eppendorf tube add 450µl N3 neutralisation buffer and invert 12 times
  5. Centrifuge for 10 minutes at 13,000 rpm
  6. Apply supernatant of the first tube from same bottle to a QIAgen Quick column
  7. Centrifuge for 1 minute and discard flow-through
  8. Apply supernatant of the second tube from same bottle to the same QIAgen Quick column
  9. Centrifuge for 1 minute and discard flow-through
  10. For each column (2 in total) add 500µl PB Wash buffer
  11. Centrifuge for 1 minute and discard flow-through
  12. For each column add 750µl of PE Final Wash
  13. Centrifuge for 1 minute and discard flow-through
  14. Centrifuge for an additional minute and discard flow-through
  15. Place column in an empty labelled eppendorf tube
  16. Add 40µl of water to the centre of the column and wait for 1 minute
  17. Centrifuge for 1 minute and place tubes on ice

I now have two bottles one names pUA66katE#A1#1 and the other pUA66katE#A1#2.

Digestions Single and Double

I will now perform a digestion to check the competency of the digestion and also to see if the plasmid contains the insert using a 2% gel.

  1. For the competency I will use the miniprep plasmid pUA66katE#A1#2, but keep 10 µl aside to run with the digest on the gel.
  2. For the insert check I will use the miniprep plasmid pUA66katE#A1#1

As this digestion is setup to test the system - I am paying extra attention to bottles and amounts. No mistake can be made!

BamHI Digestion, final 30µl

  1. 15µl pUA66katE#A1#2
  2. 0.3µl 100xBSA
  3. 3µl 10xNEB#3
  4. 1µl BamHI
  5. 11.7µl water
  6. Incubate for 2 hours at 37ºC


XhoI Digestion, final 30µl

  1. 15µl pUA66katE#A1#2
  2. 0.3µl 100xBSA
  3. 3µl 10xNEB#3
  4. 1µl XhoI
  5. 11.7µl water
  6. Incubate for 2 hours at 37ºC


Double Digestion BamHI/XhoI, final 50µl

  1. 30µl pUA66katE#A1#2
  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

Gel, insert and competency check

I prepared two gels, 1% and 2%.

  1. 1% to check the competency of the single digests
  2. 2% to check the insert

2% Gel

  1. Weigh out 1.2g Agarose powder
  2. Add 60ml 1xTAE
  3. Heat in microwave until no more specs and cool
  4. Add 2µl Ethidium Bromide
  5. Pour in prepared tray and wait until ready

1% Gel

  1. Weigh out 0.6g Agarose powder
  2. Add 60ml 1xTAE
  3. Heat in microwave until no more specs and cool
  4. Add 2µl Ethidium Bromide
  5. Pour in prepared tray and wait until ready

Loading 2%

  1. Lane 1: 10µl, 100bp NEB Quick Ladder
  2. Lane 2: 50µl, pUA66katE#A1#1 XhoI/BamHI
  3. Lane 3-8: BLANK
  4. Run at 100V, 400mA, 60 minutes

Loading 1%

  1. Lane 1: 10µl, 1kb NEB Quick Ladder
  2. Lane 2: 30µl, pUA66katE#A1#2 BamHI
  3. Lane 3: 30µl, pUA66katE#A1#2 XhoI
  4. Lane 4: 10µl, pUA66katE#A1#2 Undigested
  5. Lane 5-8: BLANK
  6. Run at 100V, 400mA, 60 minutes

Gel Picture 2% 17082010-2xdigest-pua66kate.jpg

Picture confirms that the plasmid does not contain the insert!

Gel Picture 1% 17082010-single-digest-pUA66.jpg

Picture confirms that the plasmid is digested to the correct size by XhoI and BamHI separately and further that this plasmid is self-ligating (which is not possible) or is a result of contamination e.g. an undigested plasmid or half-digested plasmid. The problem however is that the self-ligating control did not produce colonies User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Plates and this call into question how to avoid or where this issue occurs (e.g. is there contamination in the water, in the insert or other places?)

Debugging

I will now look at how to debug the vector.


Scenario

17082010-debug-f1.svg

When digesting the vector with XhoI and BamHI 4 scenarios may occur.

Type Scenario Gel
I Undigested, none of the enzymes worked Non-specific
II Single Digest - XhoI 4,260bp
III Single Digest - BamHI 4,260bp
IV Double Digest - BamHI / XhoI 4,252bp



Type II and III

17082010-debug-f2.svg

In this scenario (figure 2) the vector would simply religate and never take up the insert.

Type Ligation Gel
II XhoI + Ligation + Growth 4,260bp
III BamHI + Ligation + Growth 4,260bp


Type VI""

17082010-debug-f3.svg

In this scenario (figure 3) the vector would either produce a mega vector or take up the insert

Type Ligation Gel
IV-A Ligation +Growth resulting in a mega vector 8,504bp
IV-B Ligation +Growth resulting in a vector with the insert 4,652bp

Improbable

17082010-debug-f4.svg


In this scenario (figure 4) the vector as it has sticky ends would not be able to produce a viable plasmid.

Transformation of ligation pUA66katE from previous day

This transformation uses to overnight ligation User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/16#Ligation #2 pUA66 XhoI/BamI with katE XhoI/BamHI - overnight

  1. Collect 50µl competent cells from the -80ºC
  2. Thaw on ice for 5 minutes
  3. Add the complete 10µl of ligation reaction
  4. Wait for 5 minutes
  5. Place in water bath 42ºC for 1 minute
  6. Place back on ice for 5 minutes
  7. Add 250µl SOC
  8. Incubate in shaker for 1 hour at 37ºC
  9. Add to Kanamycin plate and spread using glassbeads
  10. Place in incubator at 37ºC overnight

Transformation of p67 plasmid

  1. Collect 50µl competent cells from the -80ºC
  2. Thaw on ice for 5 minutes
  3. Add 1µl of p67 plasmid
  4. Wait for 5 minutes
  5. Place in water bath 42ºC for 1 minute
  6. Place back on ice for 5 minutes
  7. Add 250µl SOC
  8. Incubate in shaker for 1 hour at 37ºC
  9. Add to Kanamycin plate and spread using glassbeads
  10. Place in incubator at 37ºC overnight

Overnight growth, pUA66katE

A new overnight 10ml growth was put on based on previous days transformation User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/16#Transformation, Ligation pUA66katE.

4 bottles where prepared from the 4 generated colonies

  1. 10ml LB broth
  2. Add 10µl, 50mg/µl Kanamycin
  3. Add one colony pUA66katE

Overnight growth, pBestLuc

A new overnight 10ml growth was put on based on previous days transformation User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/16#Transformation, pBestLuc

1 bottle was prepared

  1. 10ml LB broth
  2. Add 10µl, Ampicillin stock
  3. Add some colonies pBestLuc (two dense to select single colonies)