2005-06-07

Plan:
- make and image the FAM and TAMRA SEs tube
- change the lower objective to the TIRF objective
- get a fluorescence image of the 1 um^3 bead

SEs tube strands
SE1, SE2-DIAG, SE3, SE4-EE10, SE5

Fluoresceinated SE strand:
  SE3-FAM (42-mer, 436340 /M/cm @ 260nm) : FAM-CCAGAACGGCTGTGGCTAAACAGTAACCGAAGCACCAACGCT
  SE3-TAMRA

FAM
  excitation 494 nm 
  emission 518 nm

TAMRA
  excitation 542 nm
  emission 568 nm

spFRET laser emission 532 nm (green)
The filter is about 550-75 nm 
  - upper filter : (HQ575/50 M 54652)
  - lower filter : (550LP 7513)
check the type of filters ... low pass? high pass?

--> Imaging FAM labeled tube may or may not be possible due to to excitation and emission wavelengths

(8.38 am)
- the previous lower objective was replaced with the TIRF objective (see Photo_060705_001-3.jpg)
- with a blank sample (5uL of MQ H_2O in between 2 coverslips), a spot was observed on the ceiling. (see Photo_060705_002-3.jpg)
- the illuminated region is not in the center of the image .. it may be due to the small size of the focused laser.
  (see Photo_060705_004.jpg)

(9.06 am)
- now, I am preparing the 0.2 uM of SEs-TAMRA tube and a 1/20 of 1um^3 beads

2uL of 10uM SE1
2uL of 10uM SE2-DIAG
2uL of 10uM SE3-TAMRA
2uL of 10uM SE4-EE10
2uL of 10uM SE5
10uL of 10X TAE/Mg++
80uL of MQ H_2O

(10.02 am)
- a new 1/20 1um^3 bead sample was made, the dilution agent is 1X TAE/Mg++

- Rebecca (Rebecka?) said that the best imaging condition with cyber gold is with 0.1x cyber gold
  The stock in the freezer is 10,000x

(10.20 am)
- still can't align the microscope .. the alignment is tougher with 100x objective

(10.37 - 11.02 am) 
- chatting with Harry, preparing for our AFM session tomorrow
- need to find 
  Variants of #2 and #4 strands without sticky ends (REn, SEn).
    SE2-NSTK (16 mer, 164940 /M/cm @ 260 nm) : GTAGAGCACCACTGAG
    SE4-NSTK (16 mer, 155260 /M/cm @ 260 nm) : AGTTTCGTGGTCATCG
    RE2-NSTK (16 mer, 151040 /M/cm @ 260 nm) : CCTTCACACCAATACG
    RE4-NSTK (16 mer, 172660 /M/cm @ 260 nm) : GAAGATGTGGTAGTGG
  - found those strands with no problem =)

(12.50 am)
- a new sample, 1/10x 1um^3 tube was made in MQ H_2O
- finaly, could get an image using the 100x TIRF objective (see Photo_060705_005.jpg)
- note that the beam are not at the center of the black box    --> better image if the light is not straight through --> problem with the filter??
- the image was much less bright as I expected.  The setup with 40x lower objective gives a brighter image than the current setup with 100x TIRF objective.
- see Video_060705_002.3pg

(1.10 pm)
- consult Zahid for debugging the microscope
  his suggestions: 
  - check the filters
  - the instrument picks up vibration, may need to be moved to other place (my lab bench?)
- the image was slightly better with additional tweaking

(2.29 pm)
- clean up