Team:Toronto/Notebook-w09-thu
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Thursday July 14th
Thursday, 7/14
Members Present: Hamed, Kat, Bohdan, Karim, Tam, Marc
LAB:
Morning:
Afternoon:
Gel 1:
Lane 1: L1A (Ligated tetO-LacZ plate A)
Lane 2: L1B (Ligated TetO-LacZ plate B)
Lane 3: 2 log DNA Ladder
Lane 4: L2A (Ligated TetO-mCherry plate A)
Lane 5: 2B (Ligated TetO-mCherry plate B)
Lane 6: L3A (Ligated pgolB-LacZ plate A)
Lane 7: L3B. (Ligated pgolB-LacZ plate B)
Gel 2:
Lane 1: L4A (Ligated Long GolSP118A plate A)
Lane 2: L4B (Ligated LongGolSP118A plate B)
Lane 3: 2 Log DNA Ladder
Lane 4: L5A (Ligated Short TetO GolSP118A plate A)
Lane 5: L5B (Ligated Short TetO GolSP118A plate B)
Lane 6: L6A (Ligated Long GolS plate A)
Lane 7: L6B (Ligated Long GolS plate B)
Gel 3:
Lane 1: L7A (Ligate Short TetO GolS plate A)
Lane 2: L7B (Ligated Short TetO GolS plate B)
Lane 3: DNA 2-log Ladder
Lane 4: L8A (Ligated short PcrnA - LacZ plate A)
Lane 5: L8B (Ligated short PcrnA - LacZ plate B)
Lane 6: L9A (Ligated short TetO - RcnR plate A)
Lane 7: L9B (Ligated short TetO - RcnR plate B)
Summary:
Our ligation attempts were unsuccessful, as all ligated constructs appeared at the same band size (~1.7kb.) In reality, the assembled constructs should be:
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~2.9kb - L2, L4, L6
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~2.6kb - L5, L7
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~2.4kb - L1, L7, L8, L9
The ligated constructs may present to be just digested pSB1C3 backbone. We suspect that some form of re-ligation occurs between the digested fragments during our ligation protocol, due to the fact that we did not PCR purify the fragments following restriction enzyme digestion. This would mean that our UNS/cut ends re-liagate back onto our inserts instead of allowing our inserts to re-ligate with the backbone. The colonies that grow following transformation may contain some form of circularized backbone with the antibiotic resistance marker.
For future steps, we shall PCR purify all parts following RE digestion. Our only successful ligation (albeit at a larger than expected size, due to suspected PCR concatemer ligation) utilized PCR purified products (see previous L1 attempt).
Administrative:
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A P&P meeting was held with our adviser Professor Dias regarding the synthetic biology development course
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Meeting notes can be found here: https://docs.google.com/document/d/13E4ff0_HZX5zCuO1rnOHDo1tv_8cAsIg0J8OuGLRO24/edit?usp=sharing
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Add TB buffer making to our protocol.
TO DO:
For the next day:
MORNING:
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PCR amplify:
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pSB1C3 backbone
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Short mCherry
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Long linear GolSp118A
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Long linear GolS
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TetO-LacZ
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RE digest of PCR amplified parts
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PCR purification of PCR amplified and digested parts.
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Nanodrop
AFTERNOON:
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PCR amplify:
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pgolB-LacZ
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Short TetO - RcnR
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Short PcrnA - LacZ
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Short TetO-GolSP118A
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Ligation with PCR amplified, RE digested, and purified parts. Remember to use the Ligation calculator based on nanodrop results. https://docs.google.com/spreadsheets/d/1NDGf_n6F3x5Xt3SPbX-_kfOsVGvaEytrjdaVOF2fO94/edit#gid=1646133783
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Gel of all PCR amplified parts
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Transformation with ligated parts.
LAB TEAM:
LAB MANAGERS:
Purchases made today:
A | B | C | D | E | F | |
1 | Name | SKU | Unit Price | Quantity | Total (incl. tax) | Status |
2 | Colored Label Tape: 1 in. GREEN (UM) | 0007-1590110C | 4.05 | 1 | 4.58 | available |
3 | Colored Label Tape: 1 in. RED (UM) | 0005-1590110E | 4.05 | 1 | 4.58 | available |
4 | Pipette Tip 1000uL | 70.762 | 2.5 | 3 x250 | 8.47 | available |
Table1
Gmail correspondence:
Meetings/Notes:
References:
