Gel electrophoresis - PCRMar0508-2p

3% agarose (7.5 g), 25oml 0.5X TBE

Poured 7:32 am (March 6th)

Loaded 8:10 am (March 6th)

Loading order: A1-11, ladder, B1-4, 7-9, A12 (water ctrl) - Top row; C1-11, C12 (water ctrl) ladder, D1-4, 7-9, - middle row

Amt of sample loaded: 16 microlitres

Staining time: > 20min

Results: Bands for A1-3, C1-3 and B 7-9 and D 7-9.

Conclusion: Zymo DNA extracts can be used as long as a higher volume (12 microlitres) is added to the extracts. Perhaps what we need to do is clean up the Koba DNA extracts to get them to work. Zymo kit works to extract usable DNA from old leaves better than Koba, perhaps due to the filtration system for cleaning up the extracts.

 

DNA extraction L seedlings

L5 - L7′ (6 samples) extraxted to compare with original extracts:
L5 (9), L5′ (23), L6 (3), L6′ (17), L7 (21)

Protocol: ZR Plant/Seed DNA Kit

(Tip: weigh ‘empty’ tubes and then weigh after adding leaf tissue to calculate mass of tissue extracted)

Step 1: “vortex”
Used Fastprep (Bio101) FP 120, at speed 6 for 45s (highest time setting) and then speed 6.5 (highest speed setting) for 45s a couple of times. Machine started making a loud scraping sound, so I stopped it in the middle of the 3rd time at 6.5 and continued to the next step

Centrifuged at 10,000 rcf for 1 min

Step 3: L7 (21) <400, L5 (23) >400, L6 (17) <400, L5 (9) ~400, L6 (3) ~400, in step 3, there was nothing in the protocol that said to break the tip of the tube (under the filter) to allow the filtrate to pass, but this needed to be done before the tubes were put into the centrifuge.

Step 7: 2×50 for 2 samples, 1×50 + 1×14 for others, extra divided up.

Last step, did not mention that filtering “column” had other liquid in it, which added to the final volume of the DNA elutant.

N.B. Best gel pattern came from undiluted ‘Zymo’ DNA, so will test PCR without diluting.

 

Gel electrophoresis of PCRjan1508-5p3p

3% agarose, 0.5x TBE (25oml), 16 microlitres of sample

ladder: low ladder, 8 microlitre

Gel 1:
A1-12, ladder, B1-12 - top row
C1-12, ladder, D1-12 - middle row

start: 11:00 am
end: 2: 40 pm

Results: all primers worked well with FP dna samples but not with the L seedlings. See pics

Gel 2 :
E1-12, ladder, F1-12 - top row
G1-12, ladder, H1-12 - middle row

start: 10:38am
end: 1:34 pm

Results: row E had bands for only lanes 1 and 2, row F- strongs bands for 1-3, faint elsewhere, G: 1-4 strong, 4 somewhat less strong, H: 2-4-strong but4 somewhat less so. (see pics)

 

PCR Jan1508-5p3p

PCR to test L seedlings (and screen* new primers) – PCR Jan1508-5p3p

Primers: 1720/1721, 1724/25, 1728/29, 1776/77, 1778/79, 1680/ 1681*, 1718/ 1719*, 1628/ 1629*

DNA templates: SLC 28, PA 151, SM1 (positive ctrls), L1, L1’, L2, L2’, L3, L3’, L4, L4’

Taq: Biolase

start: 1:30pm End: 4:15pm

 

Gel electrophoresis of PCRjan1008-8p - 1

Gel 1-Jan 11th

Top row: A1-12, ladder, B1-12 Middle row: C1-12, ladder, D1-12

Ladder: Low ladder 8microlitresStart time: 12:22pm Start current: 41 mA Checked 1:30pm blue dye travelled ~1 cm

End : 3:55 pm (Final current: 45mA)

Gel 1 results: bands overrun. Primers with results: all except 1034/1035, but not for all DNA templates.

Gel 2-Jan 14th

Top row: E1-12, ladder, F1-12 Middle row: G1-12, ladder, H1-12 (loaded 16 microlitres of sample)

Gel 2 start: 10:57 am
End : 2:10pm

Results: bands for all 4 primers.

 

Gel electrophoresis of PCRjan0408-2p

Poured gel Jan 7th. 250ml 3% agarose, 0.5 x TBE

Loaded Jan 8th, Run at ~28 mA (slow run)

Loading order:
Top row: A1-12, ladder, B1-12, C1-2 (1180/1181)
Middle row: E1-12, ladder, F1-12, G1-2 (1150/1151)
Ladder: Low ladder (8 microlitres)

Start : 9:40am
End :
Results: few faint bands for 1180/1181, (between L8-L10)

Conclusions: screen several of the new primers with some DNA samples from FPproject then use with L samples.

Prepared 4 litres 0.5X TBE (from 5X stock made in November, 2007)

 

PCR Jan0308-2p

Primers: 1180/1181 and 1150/1151

DNA: L1-L13′ (26 samples)

A1-C2, C12: 1180/1181
D1-F2, F12:

Program: TD-cacao

Polymerase:- Biolase (0.4)

(Pour 1 gel, 30 well combs)

 

PCR dec0607-4p

Primers: 118/119 (Val’s box), 27/28 (new dil.), 35/36 (new dil. previously used), 1180/1181
Taq: BioTaq 0.4

Template: L9, L9’, L10, L10’, L12, L12’, L13, L13’ (new dilution 1/150) JA 1/21 (FP sample), CLM 90 (FP sample), JA3/11 (new extract-Dec6,07, diluted 1/100), LCT 68/S2 (new extract-Dec6,07, diluted 1/100)

JA 1/21, CLM 90 are +’ve ctrls

14x mix

 

Gel electrophoresis of PCRdec0407-1p

3% agarose gel (7.5g) poured Dec4th, 250ml 0.5x TBE

Starting current: 46 mA

Load samples @ 8:55am
Top Row: A1-12, ladder, B1-12, C1-2, F1-2,G1 (PCRdec0407-1p)
Middle Row: C12, F3-4,G3 (same pcr), Ladder, G1-8, B7-12, F11 (PCRnov3007-2p)

Run start : 9:12am
10:40(checked current @ 46mA), 11:39 (changed current from 47 to 54mA)
Run end: 12:55pm

Staining: 12:57pm-1:18pm, checked again @ 1:49pm- no improvement

Results: Bands visible only for B5-7, F3-4, G3, and very faint for samples from PCRnov3007-2p (G3,4 B7,8,12); ladder is still faint/somewhat streaky.
(F3-4, G3=JA 1/21 FPsample)

 

PCR dec0407-1p

Primer 118/119
Polymerase: Biolase (0.4 µl)

Template: L1-L13, JA 1/21 (as +’ve ctrl, old and new dilutions L-extraction and FPextraction)

F1-4=JA1/21 (7 µl)
G1,3=JA 1/21 (10 µl)