IGEM:Harvard/2007/Laboratory Notebooks/Bacterial Targeting Protocol/Week 7

Gel Purification of AIDA Digest (07/30/07)

1. Add 150 ml 1x TBS
2. Add 1.8 g of agarose
3. Heat to a boil, let cool to a warm/hot temperature.
4. Add 1.5 ul EtBr
5. Pour into gel and wait until solidified.
6. Run gel at 100-150 V

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Gel Purification of AIDA Digestl, Part 2 (07/30/07)

1.2% agarose gel, 150V Lanes:
1. 1kb+ Ladder, 10ul
2. 1kb+ Ladder, 20ul
3. blank
4. AIDA1-PCR with random library, cut with Nde1 and Not1, Kevin's reaction
5. AIDA1-PCR with random library, cut with Nde1 and Not1, Kevin's reaction
6. blank
7. AIDA1-PCR with random library, cut with Nde1 and Not1, Alex's reaction
8. AIDA1-PCR with random library, cut with Nde1 and Not1, Alex's reaction
9. blank
10. AIDA1 expression plasmid 3
11. AIDA1 expression plasmid 3
12. blank
13. AIDA1 expression plasmid 4
14. blank
15. AIDA1 expression plasmid 5
16. blank
17. AIDA1 expression plasmid 6
18. blank
19. AIDA1 expression plasmid 7

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MACS Assay - his and strep AIDA cells and his and strep OmpA1 cells with RFP (7/31/07)

Purpose: To re-test our construct and to test the OmpA loop idea.

1. OD equilibriated to 0.5
2. Followed Indirect Magnetic Labeling Protocol
   1. Used 125 ul of Strep/His and 125 ul of RFP Bacteria
   2. Used 8 ul his antibodies and 16 ul strep antibodies
   3. Used 20 ul of Beads
3. Then follow Magnetic Separation
   1. Use the additional measures

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MACS Assay results (7/31/07)

Results: There was background binding that we did not want, with about an even amount of red and white cells in both constructs. Also, the AIDA constructs grew slower as we had expected.

Conclusions: This time, we fear we may have used too small of a antibody to cell ratio, and the increased cell density would cause background binding.

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