Overall summary for 2007
First injections were on Monday, September 10, last big one was on Wednesday, September 26
Marina Freudzon and Rachael Norris were here for first week.
Two waves of things to inject, first from Mon Sept 10 to Sat Sept 16, then an apparent lull, then second wave starting Fri Sept 21, end Wed Sept 26. 

This year, we tried a lot of ways to try to improve the injection technique. Picospritzer was probably biggest advance, Jochi was advocating this strongly last summer, and we managed to get it to work this summer.
- More reliable fertilization - put concentrated sperm on ice, and made dilutions just before fertilization
- Mix siRNA with calcein so that we can tell which ones injected, potentially don't have to sort right after injections.  Development seems to be normal.
- Use higher concentration of siRNA so can inject smaller volumes
- Marina finds out that we can load multiple injections in a needle - this increases the number of embryos we can inject
- Figure out picospritzer settings so that we can inject faster using our manipulator and microscope set up

During first week, there was lots of uncertainty because there was a higher fraction of unsuccessful  phenotypes and non-development.  Not sure if this was due to injection changes.  Had to do some re-grouping to test this.

John's main interest for this year was chordin, but at first, this couldn't be repeated from last summer.  It turned out that our new mercury method was injecting less material; when we injected the same amount as in the old method, we got the result from last year.  So different siRNAs have different thresholds for giving phenotype.
Try picospritzer hooked up with Narashige SM-20 manipulator, this started to work in week 2/3.  Able to do about 100 in 2 hrs.

Picospritzer typical settings (Harvard Apparatus)
40 psi clear, 20 psi inject; 320 msec inject time
use custom glass tubing (that we use for loading capillary, 0.8 x 0.6 mm) as injection needle.  pull as long as the usual starfish injection needles.  break tip as if for mercury injection.  Back load using the filling tips from Kirschner (eppendorf physiocare concept)
8 div ~1% inject into oil calibration.
make a loading capillary with SDS for cleaning the needle 

Some ideas for next year
Keep eggs from individual females separate and screen them for injectability.
How to work in a team to increase yield.
Keep good track of siRNA amount injected into embryos.
Inject but don't sort until a few hours later; sort out the fluorescent normal cleaving embryos.
Use a fluorescent standard to double check on the amount injected by picospritzer (fluorescent drop)
Try picospritzer for screening too? 
Make several picospritzer set ups.  Fluorescence might be useful. 

-----
Daily activities

Mon, september 10
bring down microscope from 302, rachael arrives.

try multi injection with one loading - no fluorescence apparently.  try injecting without oil cap, also apparently no fluorescence

Tues, september 11
4 siRNAs injected - chordin, noggin, follistatin, FNC (41 total)
start around 8, finish around 10:30

possible improvements this year - dry sperm for more reliable fertilization.  using more concentrated siRNA in order to inject a smaller volume (easier to load pipets).  include calcein (1:1) in siRNA mixture, don't sort after injecting, just dump into dish, sort later. 

total injected  60(?)

Wed, sept 12
10 goosecoid (1:10 in calcein)
10 hex
10 brachyury
6 wnt1

total injected at end of day by all three of us   156

Thurs, september 13
nodal 2.1 - 10
frizzled 50.1 - 10 (strike) (took less than 15 min, start at 6) (load 5 at a time)
frizzled 50.2 - 12 (9 could have been strike except john came in to ask about calcein!)
(finish 6:40)
hedgehog 2 - 6 (add to Rachael's)
wnt4-1 10 (strike) finish 8:50
TTF1 -11  (9+2)
sfrp 10 (8+2)
sfrp2 10 (7+3)
bmp chordin (CB1) - 10 (strike) (11:45)
dkk - 11 (6 + 3+2) finish 12:15
total = 100

Fri, september 14
foll2 - 10 or 11.  finish 5:12 (spare)
gsc2 - 10 (strike) finish 5:26
hex2 - 10 (strike) finish 5:40
wnt134 - 10 (spare) finish 6:03
fox2    (8+ 2  ) 
restart at 7:25 after dinner at fishmonger, get animals, last 2 of fox 2
brachyury2 10 (strike) finish 7:51
dmbx2  10  poor fertilizations, threw out some by mistake etc.  finish 8:20
chordin2 6+7+6
stop 9:30
total - 89

end of day total - 635

saturday, september 15
start getting things together ~5, do chordin injections, 7:15 break for pie in sky, 8:15 return. finish 10:10

3.5 div = 30 µm diameter = 14 pl = 0.06%
5 div = 40 µm diameter = 34 pl  = 0.15%
7 div = 50 µm diameter = 65 pl =0.28%

-used fluorescence dissecting scope at different stages - before transferring from injection chamber to the dish, or after transferred to the dish.  Found that some of the ones that I thought were injected had low or no fluorescence.  In some cases, I saved the low fluorescence embryos in separate dishes.   

chordin injections (1:10 from september 11, no calcein)
0.15%  - 7 embryos
0.06% - 6 embryos
0.28%  - 5 embryos

chordin (1:10 with calcein, fresh dilution today from John)
-- all that I thought were injected were saved.  checked in the dish by fluorescence afterwards.
0.15% - 9 embryos (2 low fluorescence)
0.06% - 11 embryos  (all are low fluorescence so hard to tell)
0.28% - 6 embryos (2 low fluorescence)

noggin injections (1:10 with calcein, fresh dilution today from John)
0.15% - 9 embryos - screened by fluorescence dissecting scope before transferring to the dish.  There were 3 or 4 that had low or no fluorescence.  Didn't save these.

follistatin 1 (1:10 with calcein, fresh dilution today from John)
dish 1: 0.15% - 9 embryos (screened before transferring to dish, these were fluorescent)
dish 2: 0.15% - 3 embryos, 2 dim thought injected, 1 bright with a nubbin sticking out

BMP (fresh 1:10 with calcein)
dish 1: 0.15% - 8 embryos (screened before transferring to dish, these were fluorescent)
dish 2: 0.15% - 2 thought were injected but had low fluorescence

end of day total - 785

sunday, september 16
start around 4
try to inject without oil cap:
try small tip 2-3% tx100 with siRNA for BMP, inject at rough stage - maybe looks OK, but there were small wounds at injection site, bigger than normal
other attempts without tx100, about 1 in 3 or 4 are successful
perhaps try to leave needle in egg for 5 sec before pulling out? 
perhaps short strong burst with picospritzer would be better than hand controlled?

FGF1.1.1 - 9 embryos (reinjection at higher dose to look for expected phenotype)
ttf2 - 8 embryos (reinjection of a batch that had all dead a few days ago)

monday, september 17
work on picospritzer injection
use same settings as regular needle, they are shorter.  long needles are hard to break
unfortunately lots of embryo bursting at unexpected times, just due to penetration (not removal) of needle
ran out of nitrogen gas.  best injections were in early ruffled stage
~3 with 3.3% tx100 / BMP, ~15 with no tx100 / BMP.

Tuesday, sept 18
more picospritzer

wnt1 - 10
wnt3 - 11
chordin 1 - 7  (14 division injections)
calibrate 88 µm drop = 360 pl = 1.5%
12 div = 250 pl; 10 div = 68 µm = 160 pl   7 div = 50 µm = 65 pl
chordin 1 - 6 (10 div injections)

Thursday, sept 20
another picospritzer day.  thought was doing well, maybe 60 bra2 in 1 hr, 30 wnt1-1, but wnt1-1 looked bad after a few hours.  back to square one

friday, sept 21
start around 5:30
wnt 3-2 - ~38 (finish around 6:35) (a lot of re-adjustments back to 5 shot injections)
wnt 1-1 ~35 or so.  finish 8:10. 

Satruday, sept 22
real struggle but got it done
50 otx-1
30 brachury-2

Sunday, sept 23
10 goosecoid
11 chordin 1+2
10 admp 1+2
11 cho1 admp1
8 fox 1

started around 8:30, finish around 10:45 (50 in 2 hrs 15 min)
it took some time to get started, but then it started to go smoothly, easy "spares" after the first one.
gerhart gave me a 1.5 ml eppendorf with about 0.75 ml and piece of testes, that he broke up.  He also showed me how to see the sperm in Nikon dissecting scope with some kind of darkfield.  Couldn't do it with my Zeiss.  I pipetted directly from this, 5 ul into fertilization dish (after testing various ways to do this).  swirl dish, wait ten seconds, swirl to gather the embryos in the center for transfer
5 shot injections.  gentle entry of needle (remember "Clair de Lune" - Leon Fleischer), wait 5 seconds before pulling out.  Opened window so temperature was not 25.
Sort under dissecting scope.

Also, might be best to use needles pulled that day rather than several day old ones.

Monday, sept 24
10 bambi
10 bmp 7

picospritzer start around 7:30, end around 9:30, 135 green embryos, but not sure how many will develop correctly.  (john says 115 of ~150 the next day)

today, got two pieces of testes into 1.5 ml eppendorf, add 300 ul sea water and macerate.  Keep on ice.  Test 1 ul and find that it fertilizes close to all eggs though somewhat slowly, in chamber.  This seemed to keep the same potency for several hours. 
For picospritzer, used two batches of eggs.  The first batch tended to not lyse in the intermediate period, while the second batch did.  I noticed that the first batch was collected and not put in refrigerator, while the second was gotten from a dish in the refrigerator - could this be the cause? 

the tank leaks slowly when the picospritzer is turned on!  Maybe some setting is not quite right. 

had a couple instances of a clogged needle, only found out after inspecting the chamber afterwards.  Might be able to go faster by injecting then dumping into communal dish, rather than sorting.  The sorting could be done at a few hours for cleaving fluorescent embryos. 

Tuesday, sept 25
began with intention of doing multiple siRNA injections with picospritzer.  But this might not be that easy.  Had lots of trouble with needles.  Decided to stick to one mass injection (chordin).  In general, should try to get a strategy to get into the "zone" where injections are smooth

inject ~ 2 hrs, with some interruptions.  check on dissecting scope for clogged needle, then just dump everything into petri dish.  screen afterwards for cleaving embryos.  get 180, but I still don't know how to distinguish normal from polyspermic. 
sperm - kept on ice, add 2 ul to dish and swirl, wait 10 seconds then load.  often seeing fertilization on the slide.
As for the problem of the fragile period, I thought that it might be due to keeping eggs in the cold, but I collected eggs directly and still had the problem.  Somehow, it seems to be a problem with the needle perhaps.
Noticed - during fragile period, I seemed to get more success if the needle appears to give a very small pop from going through the fertilization envelope, and then put the needle into the egg.

~180 chordin 1/2

Wednesday sept 26
latest idea about hard to inject - maybe this is a property of the egg batch.  Perhaps could collect eggs from separate females and try injections on them, discard egg batches that are harder to inject. 

inject one fertilization round (~6) with 5 shot injections
7 from kirschner
noggin, follistatin, FNC   total ~60

chris injections with picospritzer
FGFR 10
FGF8 ~40
Fr1 ~20
Fr2 ~40

(time schedule, finish foll 5:32, myo D 5:45, pxx 5:56, Nkx, Mox 6:18, FGF8/10 6:34, Tbx, Neur, Noggin 7:05, FNC 7:21.  From myoD on, these were all single fertilization injections.  some interruptions, for pulling needles for instance.  On average though, 9 fertilizations in 1 hr 49 min (109 min) = 12 minutes per fertilization. 

thursday, sept 27
kristin constructs
1% injections without calcein (the old way!)
9 cs2 (control), 7 CMV, 8 beta actin