"

From 2010.igem.org

Contents 1 Andreas 1.1 Plasmid preparations 1.2 Clonings 1.2.1 Digestions 1.2.2 Gel verification of digestions 1.2.3 Gel extractions

Andreas

Plasmid preparations

Made plasmid preps from ON cultures.

DNA concentrations

Sample	DNA conc (ng/μl)	A260/A280
pSB1A3.BBa_J04450 (a)	329.9	1.81
pSB1A3.BBa_J04450 (b)	268.6	1.88
pSB1C3.BBa_J04450 (a)	337.8	1.89
pSB1C3.BBa_J04450 (b)	257.9	1.90
pSB1K3.BBa_J04450 (a)	131.5	1.90
pSB1K3.BBa_J04450 (b)	138.8	1.91
pEX (a)	161.3	1.92
pEX (b)	85.34	1.88
pMA.BBa_J18930 (a)	50.31	1.86
pMA.BBa_J18930 (b)	50.31	99.26
pMA.BBa_J18931 (a)	112.3	1.92
pMA.BBa_J18931 (b)	29.31	1.93
pMA.BBa_J18932 (a)	15.62	1.81
pMA.BBa_J18932 (b)	43.50	1.83

Clonings

Two types of cloning were prepared:

1. Transfer of the BBa_J1893x constructs (coding for fluorescent proteins) from pMA to pEX.
   • pEX.BBa_J1893x will be good controls when testing IPTG-induced protein expression from pEX.
2. Transfer of our pEX-carried parts to the pSB1x3 plasmids.

Digestions

The following vectors were digested with FastDigest XbaI and PstI:

• pSB1A3.BBa_J04450 (a)
• pSB1C3.BBa_J04450 (a)
• pSB1K3.BBa_J04450 (b)
• pEX (a)
• pMA.BBa_J18930 (b)
• pMA.BBa_J18931 (a)
• pMA.BBa_J18932 (b)
• pEX.SOD (prepared by Nina and Johan)
• pEX.yCCS (prepared by Nina and Johan)
• Control (pSB1A3 (b); no enzymes added)

Procedures according to digestion protocol.

2 μg of each vector was used for digestions.

Gel verification of digestions

Successful digestions were confirmed by loading 2 μ of each sample on a 1 % agarose gel:

1 % agarose, 140 V, 20 min.
Two bands for almost all samples indicated successful and complete digestions. Fragment sizes as expected. No digestion of control sample. Only one band visible for pEX.SOD and pEX.yCCS; probably due to low DNA concentrations in samples.

Gel extractions

pEX and pMA digestions were separated on agarose gels to extract BBa_J1893x, SOD and yCCS constructs, as well as the cut pEX vector. pSB1x3 digestions were not gel separated, as their insert (BBa_J04450) code for an RFP device, which allows for identification re-ligated constructs (i.e. negative clones) resulting in red colonies.

1 % agarose, 100 V, 30 min.
pEX (a), BBa_J18930 (b), BBa_J18931 (a), BBa_J18932 (b), SOD and yCCS were extracted from gel and stored in 1.5 ml Eppendorf tubes in -20°C until Monday (12/7).