Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/27 May 2015"

(Created page with "{{Template:UCLA}} =5/27/2015= ==1. VF/R Amplification of MaSp 2CA Temperature Test== *Due to lower yield of previous reactions, we decided to test lower temperatures which may g...")
 
 
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=5/27/2015=
 
=5/27/2015=
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==1. VF/R Amplification of MaSp 2CA Temperature Test==
+
==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.
 
*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.
 
*We tested amplification at 63 C, 60 C and 57 C in 25 uL reactions using the following protocol.
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|}
 
|}
 
*The reactions were prepared as a mastermix without the addition of polymerase. Reactions were split, then polymerase added indivdually.
 
*The reactions were prepared as a mastermix without the addition of polymerase. Reactions were split, then polymerase added indivdually.
−
==2. Resutls==
+
===Results===
 
*Cast 150 mL of 1.5% TAE agarose gel. Used 2 uL of NEB 50 bp ladder.
 
*Cast 150 mL of 1.5% TAE agarose gel. Used 2 uL of NEB 50 bp ladder.
 
[[File:5 27 2015.jpg|none|thumb|500px|'''Fig. 1''' Temperature test amplification of MaSp 2CA. The expected product has a size of 443 bp.]]
 
[[File:5 27 2015.jpg|none|thumb|500px|'''Fig. 1''' Temperature test amplification of MaSp 2CA. The expected product has a size of 443 bp.]]
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*There is a smear around 50 bp which may be leftover primers.
 
*There is a smear around 50 bp which may be leftover primers.
 
*We will use 63 C as annealing for future amplifications.
 
*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.
 +
{| class="wikitable"
 +
! style="font-weight: bold;" |
 +
! style="font-weight: bold;" | 2AB (112 pg/uL)
 +
! style="font-weight: bold;" | 2BC (205 pg/uL)
 +
! style="font-weight: bold;" | 2CA (219 pg/uL)
 +
|-
 +
| 5x Q5 Buffer
 +
| colspan="3" style="text-align: center;" | 81 uL
 +
|-
 +
| 10 mM dNTPs
 +
| colspan="3" style="text-align: center;" | 8.1 uL
 +
|-
 +
| 10 uM For
 +
| colspan="3" style="text-align: center;" | 22 uL
 +
|-
 +
| 10 uM Rev
 +
| colspan="3" style="text-align: center;" | 22 uL
 +
|-
 +
| Template
 +
| style="text-align: center;" | 8 uL
 +
| style="text-align: center;" | 4.3 uL
 +
| style="text-align: center;" | 4 uL
 +
|-
 +
| 5x GC Enhancer
 +
| colspan="3" style="text-align: center;" | 81 uL
 +
|-
 +
| Q5 Polymerase
 +
| colspan="3" style="text-align: center;" | 4.05 uL
 +
|-
 +
| ddH2O
 +
| style="text-align: center;" | 178.9 uL
 +
| style="text-align: center;" | 182.6 uL
 +
| style="text-align: center;" | 182.9 uL
 +
|-
 +
| style="font-weight: bold;" | Total
 +
| colspan="3" style="text-align: center; font-weight: bold;" | 405 uL
 +
|}
 +
{| class="wikitable"
 +
| 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.
 +
{| class="wikitable"
 +
! style="font-weight: bold;" |
 +
! style="font-weight: bold;" | Concentration (ng/uL)
 +
! style="font-weight: bold;" | A 260/280
 +
|-
 +
| 2AB
 +
| 233.2
 +
| 1.70
 +
|-
 +
| 2BC
 +
| 301.8
 +
| 1.67
 +
|-
 +
| 2CA
 +
| 276.4
 +
| 1.73
 +
|}

Latest revision as of 06:25, 28 May 2015

iGEM UCLA




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