Team:Warsaw/Calendar-Stage1/5 July 2010
From 2010.igem.org
(Difference between revisions)
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<br>Correct amount of plasmids, expected size. | <br>Correct amount of plasmids, expected size. | ||
<br> | <br> | ||
- | <br><div class="note"> Cloning GFP-terminator behind RBSes - approach 1 -Anderson's RBSes [ | + | <br><div class="note"> Cloning GFP-terminator behind RBSes - approach 1 -Anderson's RBSes [Ania S., Ania P., Ania O., Jarek, Kasia, Michal]</div> |
<br>1. Rescue samples from alkaline lysis no.2 by adding RNAse. | <br>1. Rescue samples from alkaline lysis no.2 by adding RNAse. | ||
- | <br>2. SpeI/PstI digest of samples 2-11 (RBSes:). time:3h | + | <br>2. SpeI/PstI digest of samples 2-11 (RBSes: |
+ | <a href="http://partsregistry.org/Part:BBa_J61100">J61100=2</a> | ||
+ | <a href="http://partsregistry.org/Part:BBa_J61101">J61101=3</a> | ||
+ | <a href="http://partsregistry.org/Part:BBa_J61107">J61107=4</a> | ||
+ | <a href="http://partsregistry.org/Part:BBa_J61117">J61117=5</a> | ||
+ | <a href="http://partsregistry.org/Part:BBa_J61127">J61127=6</a> | ||
+ | [Anderson's RBSes], | ||
+ | <a href="http://partsregistry.org/Part:BBa_B0030">B0030=7</a> | ||
+ | <a href="http://partsregistry.org/Part:BBa_B0031">B0031=8</a> | ||
+ | <a href="http://partsregistry.org/Part:BBa_B0032">B0032=9</a> | ||
+ | <a href="http://partsregistry.org/Part:BBa_B0033">B0033=10</a> | ||
+ | <a href="http://partsregistry.org/Part:BBa_B0034">B0034=11</a> | ||
+ | [community RBSes]). time:3h | ||
<br>3. XbaI/PstI digest of sample 1 (GFP+terminator). time:3h | <br>3. XbaI/PstI digest of sample 1 (GFP+terminator). time:3h | ||
<br>4. Run GFP+terminator on agarose gel [tu powinien byc zalacznik w postaci obrazka] Small amount of plasmid. | <br>4. Run GFP+terminator on agarose gel [tu powinien byc zalacznik w postaci obrazka] Small amount of plasmid. | ||
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<br>8. Transformation with the ligation mixtures. | <br>8. Transformation with the ligation mixtures. | ||
<br> | <br> | ||
- | <br><div class="note"> Cloning GFP-terminator behind RBSes - approach 2 [ | + | <br><div class="note"> Cloning GFP-terminator behind RBSes - approach 2 [Ania S., Ania P., Ania O., Jarek, Kasia |
]</div> | ]</div> | ||
<br>1. Set up overnight GFPterminator digest with XbaI/PstI. [16ul DNA from alkaline lysis no3, 1ul XbaI, 1ulPstI, 4ul BamHI buffer, water up to 40ul.] | <br>1. Set up overnight GFPterminator digest with XbaI/PstI. [16ul DNA from alkaline lysis no3, 1ul XbaI, 1ulPstI, 4ul BamHI buffer, water up to 40ul.] | ||
<br> | <br> | ||
- | <br><div class="note"> Prepare glycerol stocks from samples 1-12 [ | + | <br><div class="note"> Prepare glycerol stocks from samples 1-12 [Ania S., Ania P., Ania O., Jarek, Kasia] |
</div> | </div> | ||
- | <br>1. Set up | + | <br>1. Set up 2 ml liquid culture of each biobrick. |
Revision as of 20:59, 21 July 2010
Checking correctness of biobricks from 2010 distribution [Kasia, Jarek]
Checking: GFP-terminator=sample1, J61100=sample2, J61101=sample3, J61107=sample4, J61117=sample5, J61127=sample6 [Anderson's RBSes], B0030=sample7, B0031=sample8, B0032=sample9, B0033=sample10, B0034=sample11 [community RBSes], J23100=sample12 [synthetic promoter]
1.Finish alkaline lysis no3.
2.Run 5ul of undigested plasmids on the agarose gel.
[tu powinien byc zalacznik w postaci obrazka]
Correct amount of plasmids, expected size.
Cloning GFP-terminator behind RBSes - approach 1 -Anderson's RBSes [Ania S., Ania P., Ania O., Jarek, Kasia, Michal]
1. Rescue samples from alkaline lysis no.2 by adding RNAse.
2. SpeI/PstI digest of samples 2-11 (RBSes: J61100=2 J61101=3 J61107=4 J61117=5 J61127=6 [Anderson's RBSes], B0030=7 B0031=8 B0032=9 B0033=10 B0034=11 [community RBSes]). time:3h
3. XbaI/PstI digest of sample 1 (GFP+terminator). time:3h
4. Run GFP+terminator on agarose gel [tu powinien byc zalacznik w postaci obrazka] Small amount of plasmid.
5. Gel extraction of GFP+terminator.
6. Thermal inactivation of samples 2-6 (Anderson's RBSes) after SpeI/PstI digest.
7. Ligation of digested samples 2-6 (Anderson's RBSes) with gel-extracted GFP-terminator
8. Transformation with the ligation mixtures.
Cloning GFP-terminator behind RBSes - approach 2 [Ania S., Ania P., Ania O., Jarek, Kasia
]
1. Set up overnight GFPterminator digest with XbaI/PstI. [16ul DNA from alkaline lysis no3, 1ul XbaI, 1ulPstI, 4ul BamHI buffer, water up to 40ul.]
Prepare glycerol stocks from samples 1-12 [Ania S., Ania P., Ania O., Jarek, Kasia]
1. Set up 2 ml liquid culture of each biobrick.