Team:Baltimore/Notebook

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Notebook

Run through for abstract and scheduling:

Start with plasmid with taq gene and pst1 gene

Remove pst1 site from taq coding sequence

In order to remove the restriction site, do site directed mutagenesis (1day)
PCR
Digestion
Transformation
Screening (1day)
- Dilute DNA
- Colony PCR individually
- Digestion of PCR product with pst1
- Run gel ~2500bp

Add biobrick prefix/suffix to taq coding sequence

PCR and add the prefix and suffix as primers (one prefix has an extra AG-make sure to use the correct one) (1-2 days)
- Run gel
- Cut out of gel
Cut with restriction enzyme
Clean up DNA
Ligation with vector (could be vector with the terminator sequence, promoter and RBS)
Transformation

Add a promoter, transcriptional terminator, ribosome binding site (RBS)

Screen colonies (1 day)
- Colony PCR
- Restriction Digestion
- Clean up DNA
Sequence (1 day)

Make taq protein

Compare it to other enzymes and make sure it works

Calendar

July

Week 2: Tuesday, July 12; Wednesday, July 13; Thursday, July 14; Friday, July 15; Saturday, July 16; Sunday, July 17;

Week 3: Tuesday, July 19; Wednesday, July 20; Thursday, July 21; Friday, July 22; Saturday, July 23; Sunday, July 24; took 1 ul of each of products after npt1 digestion done last thursday, put on ice, added 25 ul of competent cells to each, shocked at 42C for 30 seconnds, placed back on ice for 1 half hour, put onto amp plates, labelled according to products 1B, 2B, 4B 5A and 5B, together with positive and negative controls (seven plates in all) into warm room. On Monday, got plates out of warm room, put into cold room late (4:00 pm)Notes for editing: addition of L when?, lost original notes put on wiki on sunday, 4B was a short draw of competent cells. What kind of competent cells? post transformation protocol, or get link for it.

Week 4: Tuesday, July 26; Wednesday, July 27; Thursday, July 28; Friday, July 29; Saturday, July 30; Sunday, July 31;

August

Week 1: Tuesday, August 2; Wednesday, August 3; Thursday, August 4; Friday, August 5; Saturday, August 6; Sunday, August 7;

Week 2: Tuesday, August 9; Wednesday, August 10; Thursday, August 11; Friday, August 12; Saturday, August 13; Sunday, August 14;

Week 3: Tuesday, August 16; Wednesday, August 17; Thursday, August 18; Friday, August 19; Saturday, August 20; Sunday, August 21;

Week 4: Tuesday, August 23; Wednesday, August 24; Thursday, August 25; Friday, August 26; Saturday, August 27; Sunday, August 28;

@@ Line 46: / Line 46: @@
 '''Week 3:'''   [[Tuesday, July 19]];   [[Wednesday, July 20]];   [[Thursday, July 21]];   [[Friday, July 22]];   [[Saturday, July 23]];   [[Sunday, July 24]];
-took 1 ul of each of products after npt1 digestion done last thursday, put on ice,  added 25 ul of l to each, shocked at 42C for 30 seconnds, placed back on ice for 1 half hour, put onto amp plates into warm room.  On Monday, got plates out of warm room, put into cold room late (4:00 pm)
+took 1 ul of each of products after npt1 digestion done last thursday, put on ice,  added 25 ul of competent cells  to each, shocked at 42C for 30 seconnds, placed back on ice for 1 half hour, put onto amp plates, labelled according to products 1B, 2B, 4B 5A and 5B, together with positive and negative controls (seven plates in all) into warm room.  On Monday, got plates out of warm room, put into cold room late (4:00 pm)Notes for editing:  addition of L when?, lost original notes put on wiki on sunday, 4B was a short draw of competent cells.  What kind of competent cells?  post transformation protocol, or get link for it.
 '''Week 4:'''   [[Tuesday, July 26]];   [[Wednesday, July 27]];   [[Thursday, July 28]];   [[Friday, July 29]];   [[Saturday, July 30]];   [[Sunday, July 31]];