Sep8-14

From 2006.igem.org

Revision as of 01:32, 9 October 2006

September 14, 2006

Andy, Natalie:

• all the I+J+E bands were roughly in the right location (base pair bracket) ~60% confidence
• ligated I+J with I13507 (made 6 replicates)

Andy, Charles:

• Made o/n of cells to be made competent
• Transformed I+J+E into 6 plates, P0456 (2005), P0152 (2005), P0352 (2005), and P0452 (2005).
• Made plates: 10 Amp, 1 Amp/Cam, 3 Amp/Kan

To-Do List:

• Make o/n growth of P0456 (2005), P0152 (2005), P0352 (2005), and P0452 (2005).
• If I+J+E colonies formed, make o/n growths at the 2 extreme temperatures and 1 at 37C
• Transform/check I+J+I plasmids.
• Make 6 Amp plates
• Make 30 competent cells
• Make o/n growth of I+J, B0015 (2005), B0031 (2005), and I13507 (2005) to make -80C freezer stock

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September 13, 2006

Digested I13507 (2005) and extracted plasmid. B = 15 ng/uL, C = 30 uL (had an extra band at 1500…so ignored it and hopefully we got the right part at ~3000)

Ligate I+J ABC with E0240 AB (2005) in all the combinations: AA, AB, BA, BB, CA, CB (part – plasmid)

To-Do List:

• Redo digestion/ligation/quantitation page
• Make more plates: Amp = 7, Amp/Kan = 2, Amp/Cyc = 1
• get following parts from the 2005 Registry: P0456 (2005) – 16 O, pSB1AC3; P0152 (2005) – 19 I, pSB1A2; P0352 (2005) – 21 E, pSB1AK3; P0452 (2005) – 23 A, pSB1AK3 and transform cells
• Transform cells with I+J+E (all 6)

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September 12, 2006

Andy, Anne, Charles, Natalie:

• Mini-prepped I+J HIJK
• Checked ABCDEFG but only ABCD worked, b/c EFG had thee bands – try again later?
• Quantitated I+J: A = 25 ng/uL; B = 15 ng/uL; C = 20 ng/uL

Andy, Elliott, Ting:

• Mini-prepped B0015 (2006) x 2, I0500 (2005) + J06801 (2006) 1:2 x 1, I0500 (2005) + J06801 (2006) 1:3 x 2.
• One I0500 (2005) + J06801 (2006) 1:2 overnight growth had no cells.

To-Do List:

• Make more plates: Amp = 7, Amp/Kan = 2, Amp/Cyc = 1
• cut I13507 (2005) with EcoRI and XbaI with Buffer 2
• get following parts from the 2005 Registry: P0456 (2005) – 16 O, pSB1AC3; P0152 (2005) – 19 I, pSB1A2; P0352 (2005) – 21 E, pSB1AK3; P0452 (2005) – 23 A, pSB1AK3 and transform cells
• ligate I+J with E0240 (2005) at a 1:3 = plasmid : insert molar ratio. And double digest to check the lengths, meanwhile, plate the cells.

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September 11, 2006

Made Mini-preps of B0015 (2005) – need to test tomorrow – and made o/n of B0015 (2005) for a freezer stock. B0015 (2006) didn’t seem to grow very well so let it grow for another day and mini-prep tomorrow.

Made many (at least 6) o/n growths of the new part I0500 + J06801

To-Do List:

• mini-prep B0015 (2006) – if cells have grown
• mini-prep all the I0500 + J06801 vials. Label them I+J
• test the mini-preps for correct lengths (I+J will have a plasmid length of pSB2K3 and a part length of 1210 + 1371 = 2581 bp long)
• if test is good, proceed to ligation with E0240 (2005) and I13507 (2005). – NOTE: need to digest the plasmid (E0240 and I13507) with EcoRI and XbaI and to digest the insert (I+J) with EcoRI and SpeI – this is a pre-insertion.
• Once ligation is complete, do an initial check to see if ligation was successful, if so, transform cells with the new parts I+J+E and I+J+I.

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September 10, 2006

Miniprepped a lot of parts:

Made an o/n growth of B0015 (2005/2006) to have miniprepped and to make freezer stock

LOOKS LIKE LIGATION WORKED!!! The initial check showed that there was a band at ~4500 (plasmid), a band at ~2000 (new part) and a band at ~1000 (old part). So, cells were transformed with the new part and plated. It seems like 1:2 molar ratio of plasmid to insert worked the best (brightest new part and faintest old part)

To-Do List:

• miniprep B0015 (2005/6) and I0500 (2005)
• prepare o/n growth of new part

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September 9, 2006

Mini-prepped everything (from yesterday) Plated B0015 (2005) and B0015 (2006) Decided to have a larger stock of mini-preps so made double amounts of o/n growth of I0500 (2005), E0240 (2005), B0031 (2005), I12006 (2005), I13507 (2006), J06801 (2006), C0056 (2005), B0034 (2005)

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September 8, 2006

Double digested parts B0015 (2005), C0056 (2005) and I13507 (2005) again with EcoRI and PstI with Buffer EcoRI. To ligate the parts, I0500 (2005) will be the host and J06801 (2006) will be the insert. The new part will then be then inserted into the E0240 (2005) host. Therefore, I0500 will be cut with SpeI/PstI, J06801 will be cut with XbaI/PstI, and E0240 will be cut with EcoRI/XbaI.

Check c0056 A (2005) again

The quantitation results are as follows:

• E0240 B, 8.0 ng/uL
• E0240 A, 6.0 ng/uL
• I0500 B, 13.5 ng/uL

To-Do List:

• transform cells with B0015 (2005)
• MP B0031 (2005), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005), I13507 (2005), J06801 (2006)
• Ligate I0500 + J06801

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