User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/19

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Contents

Checked plates

The plates I grew from yesterday User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Plasmid Transformation (pUA66katE#A1#3 and pUA66katE#A1#4) and User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Grow pUA66katE#A1 both grew. The colonies on plate pUA66katE#A1#3 looked similar to the original plate pUA66katE#A1 and indicator that this sample contains the insert. I had the digestions already running.

Gel 2%, insert screening

To screen the remaining colonies from pUA66katE#A1 ran overnight digestions on a 2% gel, User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Overnight Digestion (pUA66katE#A1#3 and pUA66katE#A1#4)

  1. pUA66katE#A1#2 Serie digested ( First XhoI for 7 hour and then BamHI for 12 hours)
  2. pUA66katE#A1#3 Double digest, XhoI/BamHI, 2 hours
  3. pUA66katE#A1#4 Double digest, XhoI/BamHI, 2 hours

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: 60µl, pUA66katE#A1#2, XhoI/BamHI
  3. Lane 3: 50µl, pUA66katE#A1#3, XhoI/BamHI
  4. Lane 4: 50µl, pUA66katE#A1#4, XhoI/BamHI
  5. Lane 5-8: Blank


Gel Picture: Image:19082010-2xdigestpUA66katE.jpg

Analysis: None of the colonies screened shows the insert. At this point I have screened all four colonies. I have detected one potential candidate but I did not take a sample and will need to redo a screening for all colonies to verify.

Prepared ladder 100bp

I prepared a new 100bp NEB ladder by adding ( where did I put this protocol?)

  1. 20µl of

Miniprep - overnight growth (67 | pUA66katE#5)

I used 2 bottles out of a total of 6 for miniprep, based on the overnight growth User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Overnight Growth (pUA66katE and plasmid 67).

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

For each of the 2 (pUA66katE#5 and p67) 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)

Digestion - (67 | pUA66katE#5)

I prepared a master mix to digest each of the 30µl plasmid to a final volume of 50µl

Master mix (total volume 40µl)

  1. 1µl 100xBSA Buffer
  2. 10µl 10xNEB#3 Buffer
  3. 2µl XhoI
  4. 2µl BamHI
  5. 25µl Water

Reaction

  1. Aliquote 20µl Master mix to each of the tubes
  2. Place in incubator at 37ºC for 2 hour

Gels 1% and 2%, vector extraction and insert screening

Prepare a 2% gel to screen for insert.

Prepare a 1% gel to cut out p67

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: 50µl of pUA66katE#5, XhoI/BamHI
  3. Lane 3-8: BLANK

Loading 1%

  1. Lane 1: 10µl of 100b NEB Ladder
  2. Lane 2: 50µl of p67, XhoI/BamHI, cut
  3. Lane 3-8: BLANK


Gel Picture 1% (extract 67) Image:19082010-67-X-B.jpg


Gel Picture 2% (pUA66katE#5, new ligation plate) Image:19082010-pUA66katE-x-b.jpg

Overnight Growth

I prepared 4 bottles of 10ml LB Broth and add 10µl of 50mg/µl Kanamycin stock.

A proper screening of the pUA66katE#A1 plate was performed by labelling each colony. As these colonies had been picked before by swabbing and now regrown I check that none of the smears overlapped.

  1. 4 Bottles were setup and I picked 4 separate and labelled both plate and tube
  2. The bottles were placed in a shaker at 37ºC overnight

SAP and Ligation

A key problem with my vector is that there seems to be non-specific binding resulting in self-ligation. This prompted me to return to the use of SAP (Shrimp Alkaline Phosphate) disrupt the binding sites and prevent self-ligation.

Some mistakes were made during this process.

My vector has a concentration of around 15ng/µl, thus in 30µl I have a total of 15ng/µl*30µl = 450ng ~ 0.5µg of DNA. The SAP is used in 1unit/µg of DNA. So I will be using 0.5µl of SAP.

SAP Reaction, final volume will be 50µl.

  1. 0.5µl SAP
  2. 30µl DNA (pUA66 XhoI/BamHI)
  3. 5µl 10xSAP Buffer
  4. 14.5µl Water
  5. Place in incubator for 15minutes at 37ºC
  6. Place in a 65ºC water bath for 15 minutes to terminate reaction

I did the same reaction for a total of 4 samples from my box.

Ligations

Four ligations were then setup using all SAP

  1. 1µl Ligation Buffer
  2. 1µl T4
  3. 50µl SAP reaction
  4. 7µl katE XhoI/BamHI

I setup two reactions in a standard ligation 1:7

  1. 1µl Ligation Buffer
  2. 1µl T4
  3. 1µl pUA66 XhoI/BamHI
  4. 7µl katE XhoI/BamHI

All ligations were incubated overnight at 37ºC


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