Team:UCLA/Notebook/Spider Silk Genetics/27 May 2015

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5/27/2015

VF/R Amplification of MaSp 2CA Temperature Test

Setup

• Due to lower yield of previous reactions, we decided to test lower temperatures which may give a higher yield.
• We tested amplification at 63 C, 60 C and 57 C in 25 uL reactions using the following protocol.

	3x Reaction Volume (uL)
5x Q5 Buffer	15
10 mM dNTPs	1.5
10 uM For	3.75
10 uM Rev	3.75
Template (110 ng/reaction)	1.5
5x GC Enhancer	15
Q5 Polymerase	0.75
ddH2O	33.75
Total	75

98 C	30 sec
98 C	10 sec
63/60/57 C	15 sec
72 C	15 sec
repeat from step 2	18x
72 C	2 min
12 C	hold

• The reactions were prepared as a mastermix without the addition of polymerase. Reactions were split, then polymerase added indivdually.

Results

• Cast 150 mL of 1.5% TAE agarose gel. Used 2 uL of NEB 50 bp ladder.

Fig. 1 Temperature test amplification of MaSp 2CA. The expected product has a size of 443 bp.

• The intensity of the bands at each temperature are approximately equal.
• For this test, we used primers reconstituted from stock. Compared to previous amplifications (5/19/2015 and 5/20/2015) the yield of amplification is improved. The old primers were probably degraded.
• There is a smear around 50 bp which may be leftover primers.
• We will use 63 C as annealing for future amplifications.

Large Scale Amplification of MaSp 2AB, 2BC, 2CA using VF/R

Setup

• Based on results above, we set up 8x 50 uL reactions for each MaSp construct using the following protocol.

	2AB (112 pg/uL)	2BC (205 pg/uL)	2CA (219 pg/uL)
5x Q5 Buffer	81 uL
10 mM dNTPs	8.1 uL
10 uM For	22 uL
10 uM Rev	22 uL
Template	8 uL	4.3 uL	4 uL
5x GC Enhancer	81 uL
Q5 Polymerase	4.05 uL
ddH2O	178.9 uL	182.6 uL	182.9 uL
Total	405 uL

98 C	30 sec
98 C	10 sec
63 C	15 sec
72 C	15 sec
repeat from step 2	18x
72 C	2 min
12 C	hold

• The protocol is written for 8.1x 50 uL reactions to give leeway in making master mix.
• There is 110 pg of template for 25 uL of reaction, which equates 220 pg in each 50 uL reaction.

Results

• PCR purified the products using Zymo Clean and Concentrator -5 Kit.
• Pooled 4x 50 uL reactions into one column, then eluted both columns using the same water each time.
• Eluted in 11 uL ddH2O.

	Concentration (ng/uL)	A 260/280
2AB	233.2	1.70
2BC	301.8	1.67
2CA	276.4	1.73