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

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Checked plates
The plates I grew from yesterday User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18 and User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18 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
 * 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:

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.


 * 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)

Gel Picture 2% (pUA66katE#5, new ligation plate)

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


 * }