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Team:Brown/Notebook/July21
From 2010.igem.org
(New page: {{:Team:Brown/templates/header}} ==Wednesday, July 21, 2010== Miniprepped the 8 overnight cultures of pGEM+WillRS ligation blue-white selection colonies (using qiagen kit) *Note - looked ...) |
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===Creation of competent cells=== | ===Creation of competent cells=== | ||
| - | 6:00PM - ODed 1mL of cells containing the | + | 6:00PM - ODed 1mL of cells containing the Lovtap reporter system #2. OD was 0.259@600nm. |
6:20 - OD was 0.285 | 6:20 - OD was 0.285 | ||
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8:40 - Put in incubator | 8:40 - Put in incubator | ||
| - | + | 9:45 - Out of incubator, spread 120ul of cells on 4 LB/Amp/Kan plates | |
| - | + | ||
| - | + | ||
Latest revision as of 23:02, 23 September 2010
Wednesday, July 21, 2010
Miniprepped the 8 overnight cultures of pGEM+WillRS ligation blue-white selection colonies (using qiagen kit)
- Note - looked at the plate today, it looks suspicious because there are blue colonies, but most are light blue (we chose the ones that were most white.
Double digest of pGEM+WillRS miniprep
20ul double digest with BamHI and Nco1
- 7.8ul dH2O
- 2ul 10x NEB buffer 3
- 8ul DNA (miniprep)
- 1ul Nco1
- 1ul BamHI
- 0.2ul BSA
20ul total volume
Incubated at 37c for 1 hour
Master mix
Made a master mix for 9 rxns:
- 70.2ul dH2O
- 18ul NEB buffer 3
- 1.8ul BSA
- 9ul Nco1
- 9ul BamHI
Nanodrop data of WillRS and pGEM (in ng/ul)
- Tube 1 - 265.5
- Tube 2 - 195.6
- Tube 3 - 164.3 (corrected from 235.3)
- Tube 4 - 129.8
- Tube 5 - 251.5 (corrected from 261.5)
- Tube 6 - 171.7
- Tube 7 - 142.0
- Tube 8 - 208.5
Test of kanamycin
7 Tubes:
- Tubes 1-5 are 50ug/ml Kan in 5ml LB with 5ul of XL1s
- Tube 6 is 5ul XL1B, no Kan
- Tube 7 is non-inoculated 5mL LB
These will be grown overnight
Gel
Cast and ran a 1% agarose gel (for the products of the recent digest). Accidentally ran for a while at 25V, this resulted in a lot of streaking, including the ladder well, so we will have to repeat the digest and gel.
Creation of competent cells
6:00PM - ODed 1mL of cells containing the Lovtap reporter system #2. OD was 0.259@600nm.
6:20 - OD was 0.285
6:48 - OD at 0.300. Placed on ice
6:58 - placed in centrifuge, spun for 5min@3k,4C
7:14 - Saw no pellet. Respun for 10min@5k,4C
7:24 - Saw pellet, added 2ml CaCl2 (forgot to resuspend!). Spun down as above
7:34 - Added 500ul CaCl2 AND RESUSPENDED!
8:08 - Added 1ul of lovtap plasmid to 2 aliquots (250ul each). Lovtap @ 105ng/ul, possibly too high, but these cells are probably not at optimum competence
8:__ - Put on ice, then heat shocked
8:40 - Put in incubator
9:45 - Out of incubator, spread 120ul of cells on 4 LB/Amp/Kan plates
