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
 * 2) pUA66katE#A1#1
 * 3) pUA66katE#A1#2
 * 4) pUA66katE#A1#3
 * 5) pUA66katE#A1#4
 * 6) 300µl 50% Glycerol
 * 7) 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
 * 2) p67, 480µl
 * 3) Place in 47ºC water bath for 10 minute, mix ever 2 minute to dissolve gel
 * 4) Add 1:1 Volume Isopropanol
 * 5) p67, 160µl
 * 6) Apply each tube to a separate column
 * 7) Centrifuge for 1 minute at 13,000 rpm, discard supernatant
 * 8) Apply remainder ( it was getting a bit full) for each tube to each related column
 * 9) Centrifuge for 1 minute at 13,000 rpm, discard supernatant
 * 10) Add 500µl QG Buffer
 * 11) Centrifuge for 1 minute at 13,000 rpm, discard supernatant
 * 12) Add 750µl PE Final wash with Ethanol
 * 13) Centrifuge for 1 minute at 13,000 rpm, discard supernatant
 * 14) Centrifuge for an additional minute at 13,000 rpm, discard supernatant
 * 15) Place columns in labelled eppendorf tubes
 * 16) Apply 30µl water to the center of each columns and wait for 1 minute
 * 17) 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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