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Team:TUDelft/14 August 2009
From 2009.igem.org
Lab Notebook
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14 August 2009
Sriram
I found that colonies grown from plates were very less (around 1 to 5). There must be something wrong with the assembly. I must run the gel on monday to check the digesion. Also i found that pLacI, pTet and pSB1C3 DNA were getting less hence I must prepare them also on monday.
Tim Weenink
First did ligation of !E and !F with the parts that were not excised from gel.
| Component for !E | volume (uL) |
| *S PCR (upstream) | 2 |
| !C PCR (downstream) | 2 |
| pSB1AK3 from !A assembly | 2 |
| T4 ligase buffer | 2 |
| T4 ligase | 1 |
| H2O | 11 |
| Component for !F | volume (uL) |
| !B PCR (upstream) | 2 |
| !D PCR (downstream) | 2 |
| pSB1AK3 from !A assembly | 2 |
| T4 ligase buffer | 2 |
| T4 ligase | 1 |
| H2O | 11 |
Then I did electroporation of the !E and !F gel extracted assemblies.
After that I did chemical transformation of !E and !F assemblies (both gel extracted and direct PCR restricted fragments)
Calin
oriTR_KO +L-ara +PCR and trbK_KO +L-ara +PCR backup plates made on 0.5x KAN.
Did miniprep on CB and CC culture tubes.
Plate 2, 4, 6, and 8 are overgrown. Cells survived electroporation. No colonies on knockout plates or control plates.
| Part | DNA concentration | 260/280 | 260/230 |
| CB | 57.7 | 2.17 | 2.30 |
| CC | 121.1 | 2.01 | 2.33 |
Ran 1.5% gel with CC and CB.
| Well | Part | Expected Plasmid Size | Status |
| 1 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | 2953 | |
| 2 | CB | 1162 | ? too light |
| 3 | CC | 311 | ? SpeI not cutting |
Digested CB with X + P (11uL DNA). Digested CC with E + S (4.5uL DNA).
Daniel
No growth although gel seems good. Let´s think about it during the weekend
