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Team:Groningen/project notebook/12 August 2011
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| + | =Fri 12 August 2011= | ||
| + | '''Vessa''' | ||
| + | ==Wet lab To Do List== | ||
| + | *Plasmid prep (back up transformans) and send for sequencing | ||
| + | **12 variants autoinducing loop | ||
| + | **RFP internal control | ||
| + | **construct 21.12 | ||
| + | |||
| + | ===Implementation=== | ||
| + | '''Result of 2nd batch transformation''' | ||
| + | <br>None of 22.xx plasmid have insert | ||
| + | <br>21.12E and 21.12F have insert --> prepare for sequencing! | ||
| + | |||
| + | ===Dry lab To Do List=== | ||
| + | *Sequences data analysis! | ||
'''Joyce''' | '''Joyce''' | ||
<br> | <br> | ||
Latest revision as of 11:56, 21 September 2011
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Fri 12 August 2011
Vessa
Wet lab To Do List
- Plasmid prep (back up transformans) and send for sequencing
- 12 variants autoinducing loop
- RFP internal control
- construct 21.12
Implementation
Result of 2nd batch transformation
None of 22.xx plasmid have insert
21.12E and 21.12F have insert --> prepare for sequencing!
Dry lab To Do List
- Sequences data analysis!
Joyce
overnight PCR was finished and a clean up has been done for the taRNA, crRNA and PRM dsDNA sequences (9 samples in total, 3 of
each). Checked clean up on agarose gel 1%. There are PCR products for taRNA 1 and 3, crRNA1-3 and PRM1-3. DNA concentrations
were low according to the nanodrop, but since the fragment is very small, the nanodrop will not give significant values.
Digestion of taRNA and pSB1A3-DT
taRNA 1 dsDNA was digested:
3μl dsDNA taRNA
1μl EcoRI
1μl SpeI
2μl FD buffer
13μl MQ
taRNA 3 dsDNA was digested:
8μl dsDNA taRNA
1μl EcoRI
1μl SpeI
2μl FD buffer
8μl MQ
pSB1A3-DT:
3μl pSB1A3-DT
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
21μl MQ
Meanwhile: plasmid prep of dsDNA plasmid overnight culture.
Ligation:
taRNA-pSB1A3-DT (sample 1 and 3 same pipetting scheme:)
8.5μl pSB1A3-DT vector
3μl dsDNA taRNA
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
5.5μl MQ water
pSB1A3-DT self ligation control:
8.5μl pSB1A3-DT vector
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
8.5μl MQ water
Transformation:
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight
