IGEM:Groningen/Notebook/iGEM 2011/2011/08/12

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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

3μl pSB1A3-DT
1μl EcoRI
1μl XbaI
3μl FD buffer
1μl FastAP
21μl MQ

Meanwhile: plasmid prep of dsDNA plasmid overnight culture.

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

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